Development of a sensitive and specific enzyme-linked immunosorbent assay for the determination of fludioxonil residues in fruit juices

Fludioxonil is a fungicide with a singular mode of action that is widely employed in the treatment of fruit crops. A competitive enzyme-linked immunosorbent assay (cELISA) has been developed and validated for the determination of residues of this fungicide in fruit juices. A collection of monoclonal antibodies (mAbs) against the fungicide fludioxonil has been produced and characterized using direct and indirect cELISAs. The high affinity achieved (IC50lower than 0.5 μg L-1) considerably improved that obtained with previous polyclonal antibodies. The proposed cELISA was applied to the analysis of fludioxonil residues in apple juice and red grape must samples with a limit of quantification of…

Enantioselective Synthesis of Cuparane Sesquiterpenes. Synthesis of (−)-Cuparene and (−)-δ-Cuparenol

(−)-Cuparene and (−)-δ-cuparenol, two cuparane-type sesquiterpenoids, were synthesized from β-cyclogeraniol in 47% and 27% overall yield, respectively, using a Katsuki−Sharpless asymmetric epoxidat...

ChemInform Abstract: Enantioselective Synthesis of Herbertane Sesquiterpenes: Synthesis of (-)-α-Formylherbertenol.

Abstract The synthesis of 4-hydroxy-3-[(1 S )-1,2,2-trimethylcyclopentyl]benzaldehyde [(−)-α-formylherbertenol 1 ], a herbertane-type sesquiterpene isolated from the leafy liverwort Herberta adunca , from β-cyclogeraniol is described. The synthesis is based on the previously described preparation of an enantiopure 1,2,2-trimethylcyclopentane synthon from which the characteristic aromatic moiety of 1 is elaborated, using a Robinson annulation and a palladium-catalysed methoxycarbonylation of an aryl triflate as key synthetic steps. The synthesis of the natural sesquiterpene (−)-α-herbertenol, also a natural sequiterpene, using the same methodology is also described.

Enantioselective Synthesis of Herbertane Sesquiterpenes. Synthesis of (−)-Herbertene and (−)-α-Herbertenol

Stereoselective synthesis of polyoxygenated atisane-type diterpenoids

Abstract A new stereoselective approach to polyoxygenated atisane-type diterpenes starting from (S)-(+)-carvone is described. The key steps involve an intramolecular Diels–Alder reaction, an unusual intramolecular diazo ketone cyclopropanation of an unsaturated ketone, and a regioselective endocyclic cleavage of a cyclopropyl carbinyl radical as key synthetic steps. The synthesis of the bioactive polyoxygenated atisanes atis-16(17)-en-3,14-dione ( 2 ) and 3R-hydroxy-atis-16(17)-en-2,14-dione ( 3 ) following this approach is presented.

Antibody generation and immunoassay development in diverse formats for pyrimethanil specific and sensitive analysis

Immunochemical techniques are complementary tools to modern analytical requirements. These methods rely on the production of immunoreagents with adequate binding properties. In the present study, a rationally designed and functionalized derivative of pyrimethanil-a modern anilinopyrimidine fungicide-was synthesized in order to generate for the first time high-affinity and selective antibodies to this xenobiotic. A single coupling procedure-based on hapten activation using N,N′-disuccinimidyl carbonate and purification of the active ester-was followed to prepare both immunizing and assay conjugates. Polyclonal antibodies were produced and characterized by enzyme-linked immunosorbent assay (E…

Hapten Synthesis and Polyclonal Antibody-Based Immunoassay Development for the Analysis of Forchlorfenuron in Kiwifruit

High-affinity polyclonal antibodies directed against the synthetic cytokinin forchlorfenuron (CPPU) were produced from three immunizing haptens with equivalent spacer arms located at different positions. A competitive immunoassay was developed with a limit of detection in buffer of 12.42 +/- 3.06 ng/L. In addition, the ability of the produced antibodies to recognize a set of synthetic CPPU analogues was studied. It was evidenced that the linker position had a strong impact on the specificity of the generated polyclonals, which were more sensitive to changes at moieties of the target analyte located furthest from the derivatization site of the immunogen. Finally, matrix effects of gold and g…

The product of a regiospecific and stereoselective ene-reaction of a polycyclic terpenoid system

The X-ray structure of di­methyl (Z)-[(8aS,2R,4aR,4bR)-8a-methoxy­carbonyl-1,4a,7-tri­methyl-8-oxo-2,3,4,4a,4b,5,8,8a,9,10-deca­hydro­phenanthren-2-yl]-2-butenedioate, C25H32O7, confirmed its relative configuration. The structure shows the bend in the molecular skeleton as found for similar compounds. Weak intermolecular C—H⋯O interactions have been found in the crystal.

Immunoassays for trifloxystrobin analysis. Part I. Rational design of regioisomeric haptens and production of monoclonal antibodies

Trifloxystrobin is one of the main active principles belonging to the strobilurin family of crop protection compounds. In this article, the synthesis of a battery of regioisomeric functionalized derivatives of trifloxystrobin is described. The same aliphatic linear carboxylated chain was introduced as spacer arm in all of the synthesized haptens, but it was located at different positions of the parent molecule. N,N′-Disuccinimidyl carbonate was employed for hapten activation, so the resulting N-hydroxysuccinimyl ester could be readily purified and efficiently coupled to proteins. After immunization and hybridoma generation, a collection of 20 mouse monoclonal antibodies from different immun…

Forchlorfenuron-mimicking haptens: From immunogen design to antibody characterization by hierarchical clustering analysis

To obtain highly-specific and selective forchlorfenuron binders, a collection of functionalized derivatives with different spacer arm locations and lengths was prepared. By immunization with target-mimicking haptens, a large battery of monoclonal and polyclonal antibodies against this synthetic cell regulator was produced and exhaustively characterized in two immunoassay formats using homologous and heterologous conjugates. Antibodies with IC50 values lower than 0.3 nM were successfully raised from the prepared immunogens, thus evidencing the efficacy of the explored strategies. In order to identify significant epitopes in the antibody-antigen interaction, a series of new chemical forchlorf…

Synthesis of oxygenated spongiane-type diterpenoids from carvone

Abstract A new diastereoselective approach to oxygenated spongiane diterpenes starting from ( R )-(−)-carvone is described. The carvone is incorporated as the B ring in the final spongiane framework using a B→AB→ABC→ABCD approach, which involves an intramolecular Diels–Alder reaction and the regioselective ring-opening of a dihydrofuran ring as key synthetic steps. The structure of the key intermediate in this approach has been verified by X-ray crystallography.

Assessment of the Optimum Linker Tethering Site of Alternariol Haptens for Antibody Generation and Immunoassay Development

Immunochemical methods for mycotoxin analysis require antigens with well-defined structures and antibodies with outstanding binding properties. Immunoreagents for the mycotoxins alternariol and/or alternariol monomethyl ether have typically been obtained with chemically uncharacterized haptens, and antigen conjugates have most likely been prepared with mixtures of functionalized molecules. For the first time, total synthesis was performed, in the present study, to obtain two haptens with opposite linker attachment locations. The functionalized synthetic haptens were purified and deeply characterized by different spectrometric methods, allowing the preparation of bioconjugates with unequivoc…

Concise and modular synthesis of regioisomeric haptens for the production of high-affinity and stereoselective antibodies to the strobilurin azoxystrobin

The immune response to regioisomeric haptens of azoxystrobin with varied derivatization sites was studied. Based on the Sonogashira and Suzuki-Miyaura couplings and following a straightforward modular design, we have synthesized four haptens with the same linker anchored through C-C bonds and located at different sites of the molecule. The most stereoselective antibodies were produced from immunogens with the spacer arm at a distal position from the β-methoxyacrylate moiety characteristic of strobilurins. Moreover, we observed that assay cross-reactivity was reliant on the functionalization site of the competitor derivative. Finally, the antibody binding site was explored using synthetic ch…

Development of competitive enzyme-linked immunosorbent assays for boscalid determination in fruit juices

Abstract Boscalid is a modern, broad-spectrum carboxamide pesticide highly efficient against most fungal diseases affecting valuable crops. In this study, a boscalid-mimicking derivative with a six-carbon spacer arm replacing the chlorine atom at the pyridine ring of the target molecule was synthesized and coupled to carrier proteins. Following rabbit immunization, antibodies against this agrochemical were obtained for the first time, and they were characterised in terms of affinity and specificity, tolerance to solvents, and robustness to changes in buffer pH and ionic strength, using two assay formats. Both of the optimised immunoassays showed limits of detection below 0.1 μg/L. Moreover,…

Immunoreagent generation and competitive assay development for cyprodinil analysis.

Cyprodinil is an anilinopyrimidine fungicide applied worldwide for the prevention and treatment of highly destructive plant diseases in a large variety of crops, including cereals, fruits, and vegetables. This paper describes the development of the first reported immunoassays for cyprodinil. Two original haptens have been synthesized and conjugated to different carrier proteins, and polyclonal antibodies have been produced. Moreover, competitive enzyme-linked immunosorbent assays have been developed and characterized for the analysis of this widely used pesticide. The influence of organic solvents and buffer conditions over the assay analytical parameters was studied. The IC 50 values of th…

Rational design of a fluopyram hapten and preparation of bioconjugates and antibodies for immunoanalysis

A fluopyram hapten was designed in which insignificant electronic and structural modifications were foreseen and all potentially interacting chemical moieties were maintained. This hapten was prepared by total synthesis and three immunologically active bioconjugates were obtained and characterized. High-affinity and specific antibodies to fluopyram were raised.

CONVERSION OF RESIN ACIDS INTO STEROIDAL COMPOUNDS. A REVIEW

(1991). CONVERSION OF RESIN ACIDS INTO STEROIDAL COMPOUNDS. A REVIEW. Organic Preparations and Procedures International: Vol. 23, No. 3, pp. 321-356.

Hapten Design and Antibody Generation for Immunoanalysis of Spirotetramat and Spirotetramat-enol

Spirotetramat - a tetramic acid insecticide - is rapidly metabolized or degraded to give spirotetramat-enol; so, common residue definitions include the sum of both compounds. In the present study, two spirotetramat-functionalized derivatives (haptens) have been designed to generate immunoreagents to these molecules for rapid immunochemical analysis. Haptens have been synthesized with alternative linker tethering sites and, for the first time, high-affinity antibodies have been generated with different specificities to these active principles. Two sensitive assays have been developed using the same antibody in different formats, and by using linker-site heterologous haptens, the selectivity …

Moiety and linker site heterologies for highly sensitive immunoanalysis of cyprodinil in fermented alcoholic drinks

Cyprodinil is a new-generation anilinopyrimidine fungicide widely used in crop protection and frequently found in fruits. In this study, novel derivatives of cyprodinil with linker site heterologies were synthesized and employed in order to produce antibodies with enhanced affinity. Moreover, moiety-heterologous haptens were designed and prepared for assay sensitivity improvement. Two competitive enzyme-linked immunosorbent assays for the analysis of this active substance were developed using direct and indirect formats, achieving IC50 values around 0.15 μg/L. Analytical figures of merit and usability of the optimized assays were evaluated with wine and cider as model food processed matrice…

Ready Access to Proquinazid Haptens via Cross-Coupling Chemistry for Antibody Generation and Immunoassay Development.

17 pages, 4 tables, 5 figures.-- Publisher's PDF

Rationally designed haptens for highly sensitive monoclonal antibody-based immunoanalysis of fenhexamid.

Immunochemical methods have been consolidated during the last few years as complementary analytical strategies for chemical contaminant and residue determination. However, generation of suitable immunoreagents for small organic molecules demands adequate hapten design. In this study, fenhexamid was considered as a model compound and novel haptens were designed and synthesized in order to evaluate the influence of the linker tethering site on antibody binding properties and immunoassay parameters. Haptens were conceived with the spacer arm at different positions, while the more antigenic aromatic moiety was kept free. The synthesis of these functionalized compounds was accomplished by total …

Synthetic studies on the preparation of oxygenated spongiane diterpenes from carvone

Abstract The paper describes a new diastereoselective approach to oxygenated spongiane diterpenes functionally related to natural dorisenones. The strategy followed for the preparation of the spongiane framework, a B→AB→ABC→ABCD approach, is based on the preparation of epoxydecalone 11 (AB rings) from R-(−)-carvone, followed by an intramolecular Diels–Alder reaction for the construction of the C ring (compound 26 ). Further manipulation of the Diels–Alder adduct functionality allows the completion of the spongiane framework and the elaboration of several oxygenated spongiane-type compounds. The structures of two compounds 27 and 31 , has been established by single-crystal X-ray crystallogra…

Combined heterologies for monoclonal antibody-based immunoanalysis of fluxapyroxad

Nowadays, instrumental methodologies and rapid bioanalytical techniques complement each other for the analysis of toxic chemical compounds. Fluxapyroxad was commercialized a few years ago as a fungicide and today it is being used worldwide to control a variety of pests. In the present study, the development of monoclonal antibody-based immunochemical methods for the analysis of this chemical in food samples was evaluated for the first time. Novel haptens were synthesized and protein bioconjugates were prepared. High-affinity and specific monoclonal antibodies to fluxapyroxad were generated from two haptens with alternative linker tethering sites. Haptens with linker site heterology and a st…

Monoclonal antibody-based immunoassays for cyprodinil residue analysis in QuEChERS-based fruit extracts

Cyprodinil is among the most common agrochemical residues found in highly perishable fruits, such as strawberries. In the present study, high-affinity monoclonal antibodies to this anilinopyrimidine fungicide were raised for the first time with the aim to produce valuable immunochemical analytical assays. Cyprodinil bioconjugates and the generated novel monoclonal antibodies were employed for sensitive competitive immunoassay development in two different formats. The limits of detection of the optimized assays were 20 and 30 ng L-1 for the indirect and direct assay, respectively. Influence over assay parameters of different physicochemical factors was studied. Strawberry samples were extrac…

Immunochemical rapid determination of quinoxyfen, a priority hazardous pollutant

In 2013, quinoxyfen was included in the list of priority hazard pollutants of the European Water Framework Directive due to its toxicity to aquatic organisms. However, few analytical methods for the analysis of this fungicide have been reported and no rapid immunochemical methods have been published so far. In the present study, immunoreagents for quinoxyfen analysis were generated for the first time and an enzyme-linked immunosorbent assay was developed. Two carboxylated derivatives of quinoxyfen were designed on the basis of the minimum energy conformation of the target compound. Active esters of those novel compounds were prepared using N,N′-disuccinimidyl carbonate, and purified for cov…

Immunoanalytical methods for ochratoxin A monitoring in wine and must based on innovative immunoreagents.

Immunochemical methods are highly deployed in analytical laboratories worldwide for monitoring the incidence of mycotoxins in the food chain. Nevertheless, most conventional immunoassays for ochratoxin A (OTA), including commercial kits, show limitations to robustly determine this mycotoxin in grape-derived products below regulated levels (2 ng/mL). Herein, two rapid tests for sensitive OTA determination in wine and must were developed capitalizing on a collection of bioconjugates from innovative synthetic haptens and monoclonal antibodies with subnanomolar affinity. The ELISA (LOD = 8 pg/mL) showed excellent performance in recovery studies, and it was applied to survey commercial wines and…

1H,13C and19F NMR spectroscopy of polyfluorinated ureas. Correlations involving NMR chemical shifts and electronic substituent effects

Seventeen N-(mono-, di-, tri-, tetra- and penta-fluorophenyl)-N′-(3-nitrophenyl)ureas were prepared and characterized. Complete assignment of their 1H, 13C and 19F NMR data was undertaken and the correlation of the chemical shifts of the ureido protons with field-inductive and mesomeric electronic substituent parameters was studied using the Swain–Lupton model. The best correlations were obtained when the study was limited to certain substitution patterns, e.g. non-ortho, mono-ortho- and di-ortho-fluorinated ureas, which reveal probable changes in conformations caused by the degree of ortho fluorination at the phenyl ring. Additionally, there is an excellent linear cross-correlation between…

An approach to erythrophleum alkaloids. Synthesis of methyl (−)-4-epi-cassamate

Abstract A synthetic approach to Erythrophleum alkaloids, involving as a key-step the regiospecific deprotonation of the ketone 5a, is described.

Generation of anti-azoxystrobin monoclonal antibodies from regioisomeric haptens functionalized at selected sites and development of indirect competitive immunoassays

Azoxystrobin is a modern strobilurin fungicide used around the world to combat prime diseases affecting highly valuable crops. Accordingly, residues of this chemical are frequently found in food, even though mostly under maximum tolerated levels. We herein describe the development of an indirect competitive immunoassay for the determination of azoxystrobin residues. A panel of monoclonal antibodies displaying subnanomolar affinity to azoxystrobin was generated using, as immunizing haptens in mice, four functionalized derivatives carrying the same spacer arm located at different rationally chosen positions. This collection of antibodies was thoroughly characterized with homologous and hetero…

Synthesis of fluorinated drimanes. Preparation of 9αF-drimenin

Abstract A stereoselective approach to the 9α-fluorinated analogue of the natural drimane sesquiterpene drimenin starting from β-ionone is described. β-Ionone is transformed into a bicyclic β-cetoester from which 9αF-drimenin is prepared through stereoselective electrophilic fluorination at the C-9 with N -fluorobenzenesulfonimide followed by Wittig methylenation, allylic bromination, bromine-hydroxyl exchange and γ-lactonization.

Stereostructural revision of auricularic acid synthesis of 4-epi-auricularic acid

Abstract Cleistanth-13,15-dien-18-oic acid (1) has been synthesised from methyl 13-keto-podocarp-8(14)-en-18-oate (3). It is demonstrated that auricularic acid is an epimer at C-4 of 1, so it must be reformulated as cleistanth-13,15-dien-19-oic acid (2).

Preparation and Promotion of Fruit Growth in Kiwifruit of Fluorinated N-Phenyl-N‘-1,2,3-thiadiazol-5-yl Ureas

Seventeen phenyl-fluorinated analogues of thidiazuron [N-phenyl-N'-(1,2,3-thiadiazol-5-yl)urea, TDZ] have been prepared and characterized. The effects of each fluorinated urea on growth and quality of kiwifruits (Actinidia deliciosa) were evaluated by comparison with untreated (control) and TDZ-treated fruits. The results obtained showed a clear dependence of the growth-promoting activity of these fluorinated ureas on the pattern and degree of fluorine substitution in the phenyl ring. The most effective for promoting fruit growth was N-(2,3,5,6-tetrafluorophenyl)-N'-(1',2',3'-thiadiazol-5'-yl)urea at 25 ppm (at harvest, treated fruits were 58% heavier than untreated ones) followed by N-(3,5…

Monoclonal antibodies with subnanomolar affinity to tenofovir for monitoring adherence to antiretroviral therapies: from hapten synthesis to prototype development

Approximately 32 million people have died of HIV infection since the beginning of the outbreak, and 38 million are currently infected. Among strategies adopted by the Joint United Nations Programme on HIV/AIDS to end the AIDS global epidemic, the treatment, diagnosis, and viral suppression of the infected subjects are considered crucial for HIV prevention and transmission. Although several antiretroviral (ARV) drugs are successfully used to manage HIV infection, their efficacy strictly relies on perfect adherence to the therapy, which is seldom achieved. Patient supervision, especially in HIV-endemic, low-resource settings, requires rapid, easy-to-use, and affordable analytical tools, such …

Preparation of 9a-Fluorinated Sesquiterpenic Drimanes and Evaluation of Their Antifeedant Activities

19 pages, figures, and tables statistics.

Sensitive Monoclonal Antibody-Based Immunoassays for Kresoxim-methyl Analysis in QuEChERS-Based Food Extracts

Kresoxim-methyl is nowadays widely used to combat a diversity of common diseases affecting high-value crops. In this article, we report the development and characterization of two novel immunoassays for the analysis of this pioneer strobilurin fungicide, and for the first time, a validation study with food samples was performed. A direct and an indirect competitive immunoassay based on a new anti-kresoxim-methyl monoclonal antibody were developed for sensitive and specific chemical analysis. Optimized assays showed limits of detection of 0.1 μg/L. Fruit and vegetable samples were extracted with acetonitrile by the QuEChERS procedure and analyzed by the developed immunoassays after a simple …

Enzyme and lateral flow monoclonal antibody-based immunoassays to simultaneously determine spirotetramat and spirotetramat-enol in foodstuffs.

Spirotetramat is employed worldwide to fight insect pests due to its high efficiency. This chemical is quickly metabolized by plants into spirotetramat-enol, so current regulations establish that both compounds must be determined in foodstuffs for monitoring purposes. Nowadays, immunochemical methods constitute rapid and cost-effective strategies for chemical contaminant analysis at trace levels. However, high-affinity binders and suitable bioconjugates are required. In this study, haptens with opposite functionalisation sites were synthesized in order to generate high-affinity monoclonal antibodies. A direct competitive enzyme-linked immunosorbent assay with an IC50 value for the sum of sp…

An Efficient Stereoselective Synthesis of Stypodiol and Epistypodiol

An efficient synthesis of stypodiol (1) and its epimer at C-14, epistypodiol (2), was accomplished starting from (S)-(+)-carvone (7). The synthesis of both epimeric compounds proceeds through common intermediates using an IMDA reaction, a sonochemical Barbier reaction, and an acid-catalyzed quinol−tertiary alcohol cyclization as key synthetic steps.

Protein-Free Hapten-Carbon Nanotube Constructs Induce the Secondary Immune Response

Carbon nanotubes are novel technological tools with multiple applications. The interaction between such nanoparticles and living organisms is nowadays a matter of keen research by academic and private institutions. In this study, carbon nanotube constructs were investigated as delivery vehicles for immunostimulation and induction of the secondary immune response to a small organic molecule, namely, a hapten. Two types of nanoconstructs were prepared: on one hand, carbon nanotubes carrying a protein bioconjugate of a hapten covalently linked to the carbon surface, and on the other hand, covalent carbon nanotube constructs of the same model chemical compound without the carrier protein. Nanot…

Regiospecific and stereoselective ene reaction of the A-ring methylcyclohexene moiety of polycyclic terpenoid systems with dimethyl acetylenedicarboxylate

Polycyclic terpenoid compounds with a methylcyclohexene moiety at the A-ring, such as 1 and 7, give a regio- and stereoselective ene reaction when heated at low temperatures with dimethyl acetylenedicarboxylate. The structure and stereochemistry of the compound formed in the case of 1, e.g. 5, is determined by X-ray analysis.

Click chemistry-assisted bioconjugates for hapten immunodiagnostics

Bioorthogonal reactions have revolutionized the way low molecular weight compounds are coupled to biomolecules. Organic chemistry, polymer science, and chemical biology are among the disciplines that are benefited the most from this breakthrough. Despite the reliability of the click chemistry concept for the efficient and chemoselective functionalization of biomacromolecules with haptens at preferred positions, the fact that azide–alkyne cycloaddition reactions originate new chemical moieties as part of the linker may have delayed their application in the immunodiagnostic field. Using the mycotoxin ochratoxin A as a model compound, we herein demonstrate for the first time that bioconjugates…

Hapten synthesis, monoclonal antibody generation, and development of competitive immunoassays for the analysis of picoxystrobin in beer.

Abstract This paper describes the original synthesis of a functionalized derivative of the fungicide picoxystrobin and the generation of the first reported monoclonal antibodies against this strobilurin pesticide. The synthetic hapten was prepared by total synthesis from commercial chemicals and incorporating the spacer arm through a carbon–carbon single bond. Also, to obtain the immunogen, an uncommon hapten activation strategy based on N,N′-disuccinimidyl carbonate was employed, affording high activation yields and clean and reproducible coupling results. With these immunoreagents, two enzyme-linked immunosorbent assays (ELISAs) were developed: a competitive one-step assay using the antib…

Mepanipyrim haptens and antibodies with nanomolar affinity

Mepanipyrim is an anilinopyrimidine fungicide used worldwide for crop protection. With the aim of developing useful immunoreagents for mepanipyrim immunoanalysis, two new functionalized derivatives were prepared and antibodies were generated. Affinity and specificity were assessed by direct and indirect competitive ELISA using homologous and heterologous conjugates. Although all antibodies were selective for the target analyte, the immunizing hapten structure was revealed as a determinant for high-affinity antibody production (IC50 = 3 nM). © 2013 The Royal Society of Chemistry.

Development of monoclonal antibody-based competitive immunoassays for the detection of picoxystrobin in cereal and oilseed flours

Picoxystrobin is a new generation fungicide primarily developed to be used in cereal crops. In the present study a novel collection of specific monoclonal antibodies has been produced using different immunizing haptens based on a carboxy-functionalized identical spacer arm attached to alternative positions of the target pesticide molecule. Two competitive enzyme-linked immunosorbent assays have been developed employing hapten heterology, one using the antibody-coated direct format and the other in the conjugate-coated indirect format. Both immunoassays have been characterized in terms of selectivity, solvent tolerance, and buffer conditions, affording similar limits of detection at or below…

Exploring alternative hapten tethering sites for high-affinity anti-picoxystrobin antibody generation

The relevance of the linker tethering site in haptens was investigated for antibody generation and immunoassay development. Three derivatives of the strobilurin fungicide picoxystrobin were synthesized with the same functionalized spacer arm located at three different positions. Protein conjugates of those haptens were employed as immunogens, and novel polyclonal antibodies were produced and characterized. All haptens afforded highly specific antibodies, but different affinities to the free analyte were observed among the obtained antisera. Next, competitive enzyme-linked immunosorbent assays were studied in several formats, and site heterology was confirmed as an effective strategy for det…

Fluxapyroxad Haptens and Antibodies for Highly Sensitive Immunoanalysis of Food Samples

Fluxapyroxad is a new-generation carboxamide fungicide, with residues increasingly being found in food samples. Immunochemical assays have gained acceptance in food quality control as rapid, cost-effective, sensitive, and selective methods for large sample throughput and in situ applications. In the present study, immunoreagents to fluxapyroxad were obtained for the first time, and competitive immunoassays were developed for the sensitive and specific determination of fluxapyroxad residues in food samples. Two carboxyl-functionalized analogues of fluxapyroxad were prepared, and antibodies with IC50 values in the low nanomolar range were generated from both haptens, though a dissimilar respo…

ChemInform Abstract: Stereoselective Construction of the Tetracyclic Scalarane Skeleton from Carvone.

The tetracyclic scalarane skeleton 22 has been constructed from (S)-(+)-carvone using two intramolecular Diels–Alder reactions as key synthetic steps.

General Diastereoselective Synthetic Approach toward Isospongian Diterpenes. Synthesis of (−)-Marginatafuran, (−)-Marginatone, and (−)-20-Acetoxymarginatone

This work describes a synthetic approach to the carbocyclic skeleton of isospongian diterpenes that uses the commercially available monoterpene (S)-carvone as a C-ring synthon, which is incorporated into the tetracyclic isospongian framework via a C -> ABC -> ABCD ring annulation strategy using intramolecular Diels-Alder and ring-closing metathesis reactions. This approach has been successfully used to prepare both the title natural isospongians and several nonnatural oxygenated analogues. A preliminary evaluation of the inhibitory activity of the small collection of synthesized isospongians on the mammalian mitochondrial respiratory chain revealed that most were able to inhibit the integra…

ChemInform Abstract: Regiospecific and Stereoselective Ene Reaction of the A-Ring Methylcyclohexene Moiety of Polycyclic Terpenoid Systems with Dimethyl Acetylenedicarboxylate.

Synthesis of azoxystrobin transformation products and selection of monoclonal antibodies for immunoassay development

The use of agrochemicals for crop protection may result in the presence of toxic residues in soils and aquatic environments, besides in foodstuffs. Most often just the parent compound is included in the definition of pesticide residue, even though chemicals resulting from biotransformation and degradation routes might also be of toxicological relevance. Azoxystrobin is a broad-spectrum systemic fungicide widely used worldwide to combat pathogenic fungi affecting plants. We herein report the synthesis and detailed chemical characterization of several of the most relevant metabolites and degradates of azoxystrobin. These compounds were further employed as ligands for screening a collection of…

Immunochemical method for penthiopyrad detection through thermodynamic and kinetic characterization of monoclonal antibodies.

Immunoassays are nowadays being employed for rapid contaminant analysis in clinical, environmental, and agrochemical samples. A thorough characterization of the antibody‒antigen interaction can bring light to the immunoreagent selection process in order to develop sensitive and robust tests. Thus, determination of equilibrium and reaction rate constants is usually recommendable. However, this can be quite tricky for low molecular weight compounds, and competitive strategies are commonly followed to estimate apparent affinity values. In the present study, a collection of monoclonal antibodies to penthiopyrad was raised for the first time, and apparent equilibrium constants were assessed by t…

Highly sensitive monoclonal antibody-based immunoassays for boscalid analysis in strawberries

Boscalid is an agrochemical recently developed for crop protection and the most significant member of the succinate dehydrogenase inhibitor group of fungicides. In this study, a collection of high-affinity monoclonal antibodies was generated to boscalid. By using a series of haptens with a linker at alternative tethering sites of the boscalid framework, specific antibodies were isolated as well as antibodies that also recognized the main boscalid metabolite. Two immunoassays were developed using different ELISA formats. Optimized assays displayed very high sensitivities (limits of detection were near 0.01 µg/L). Trueness and precision for the determination of the target analyte in strawberr…

Synthetic Haptens and Monoclonal Antibodies to the Cyanotoxin Anatoxin‐a

Early warning systems for monitoring toxic events may benefit from the availability of monoclonal antibodies enabling the sensitive and specific detection of anatoxin-a, a cyanotoxin involved in numerous cases of animal poisoning resulting from toxic algal blooms in freshwaters. Through the synthesis of three functionalized derivatives of anatoxin-a, we have succeeded in generating the first-ever reported immunoreagents (bioconjugates and antibodies) suitable for the development of immunoanalytical approaches aimed at rapid and onsite detection of this harmful cyanotoxin.

Immunoreagents and competitive assays to fludioxonil

Fludioxonil is a new-generation fungicide widely used for postharvest fruit protection. The aim of this study was to produce hitherto unreported immunoreagents for Fludioxonil analysis by immunoassay. Derivatives of this agrochemical were synthesized with different linker tethering sites. Those functionalized haptens were activated, and the purified active esters were efficiently conjugated to different carrier proteins for immunogen and assay antigen preparation. Antibodies to Fludioxonil were raised in rabbits, and their selectivity and affinity were characterized, revealing the significance of the linker. Those antibodies were evaluated using homologous and heterologous conjugates by dir…

Haptens, bioconjugates, and antibodies for penthiopyrad immunosensing

4 pages, 1 table, 2 figures.

Site-heterologous haptens and competitive monoclonal antibody-based immunoassays for pyrimethanil residue analysis in foodstuffs

Pyrimethanil is a widely used post-harvest fungicide whose residues are commonly found in fruits and vegetables. We herein report the synthesis of two new functionalized derivatives of pyrimethanil carrying an equivalent spacer arm at different positions of the molecule. The influence of the linker tethering site on the affinity of monoclonal antibodies was shown. Moreover, the development of competitive immunoassays in different formats and with high sensitivity to the target analyte - IC50 values below 0.3μg/L - is described. Optimized assays were characterized by the determination of the limit of quantification, trueness, and precision using water-diluted QuEChERS extracts of fruits and …

Diastereoselective synthesis of antiquorin and related polyoxygenated atisene-type diterpenes

Abstract A diastereoselective approach to polyoxygenated atisene-type diterpenes starting from (S)-(+)-carvone is described. The key steps used in the preparation of the atisene framework are an intramolecular Diels–Alder reaction, an intramolecular diazoketone cyclopropanation, an endocyclic cyclopropane ring cleavage and the regioselective reduction of an allylic bromide by a low-valent chromium species. The synthesis of natural atisanes antiquorin (1), atis-16-ene-3,14-dione (3), atis-16-ene-2,3,14-trione (8) and 3β-hydroxy-atis-16-ene-2,14-dione (9) following this approach is presented. Also described is the synthesis of 18-hydroxy-atis-16-ene-3,14-dione (5), the structure erroneously a…

Fungicide multiresidue monitoring in international wines by immunoassays

24 pages, 5 figures, 1 table. -- Available online 10 November 2015.

Synthetic Haptens and Enantioselective Monoclonal Antibodies to the Cyanotoxin Anatoxin‐a

Early warning systems for monitoring toxic events may benefit from the availability of monoclonal antibodies enabling the sensitive and specific detection of anatoxin‐a, a cyanotoxin involved in numerous cases of animal poisoning due to toxic algal blooms in freshwaters. Through the synthesis of three functionalized derivatives of anatoxin‐a, we have succeeded in generating the first‐ever reported immunoreagents (bioconjugates and antibodies) suitable for the development of immunoanalytical approaches aimed at rapid and onsite detection of this harmful cyanotoxin.

A unified synthetic approach to trachylobane-, beyerane-, atisane- and kaurane-type diterpenes

A general synthetic approach to the polycyclic carbon skeleton of biogenetically related trachylobane, beyerane, atisane, and kaurane diterpenes from carvone is described. The skeleton of these diterpenes is prepared from a common intermediate, that is, 25, readily prepared from carvone using an IMDA reaction and an intramolecular diazo ketone cyclopropanation of an unsaturated ketone as key steps. The tetracyclic diterpene ring systems are obtained from this key trachylobane-type intermediate through the regioselective reductive cleavage of the cyclopropane ring, after adequate modification of the functionalization around the tricyclo[3.2.1.02,7]octane moiety.

ChemInform Abstract: Conversion of Resin Acids into Steroidal Compounds. A Review

(1991). CONVERSION OF RESIN ACIDS INTO STEROIDAL COMPOUNDS. A REVIEW. Organic Preparations and Procedures International: Vol. 23, No. 3, pp. 321-356.

Synthesis of site-heterologous haptens for high-affinity anti-pyraclostrobin antibody generation.

The design and synthesis of functional chemical derivatives of small organic molecules is usually a key step for the intricate production of a variety of bioconjugates. In this respect, the derivatization site at which the spacer arm is introduced in immunizing conjugates constitutes a highly critical parameter for the generation of high-affinity and selective antibodies. However, due to the usual complexity of the required synthetic procedures, the appropriate comparison of alternative tethering positions has often been neglected. In the present study, meticulous strategies were followed to prepare synthetic derivatives of pyraclostrobin with the same linkers located at diverse rationally-…

Syntheses of oxygenated spongiane diterpenes from carvone. Synthesis of dorisenone C

Abstract The synthesis of dorisenone C, a representative member of the spongiane-type diterpene family, is described. The synthesis follows a B→AB→ABC→ABCD approach and is based on the initial preparation of the previously known hydroxy-aldehyde 14 (AB rings) from R -(−)-carvone, followed by an intramolecular Diels–Alder reaction between an oxygenated diene moiety and an acetylenic dienophile for the construction of the C ring (compound 22 ), and adequate manipulation of the Diels–Alder adduct functionality for completion of the spongiane framework.

ChemInform Abstract: New Route to Herbertanes via a Suzuki Cross-Coupling Reaction: Synthesis of Herbertenediol.

Abstract The synthesis of herbertenediol, a relevant member of the herbertane-type sesquiterpene family, is described. The synthesis is based on a new general approach to this group of sesquiterpenes where the herbertane skeleton is constructed using a Suzuki cross-coupling reaction and a [2,3]-sigmatropic Still–Wittig rearrangement as key synthetic steps.

Structure-immunogenicity relationship of kresoxim-methyl regioisomeric haptens

Kresoxim-methyl was one of the two first strobilurins to be discovered, and nowadays it is widely used as an antifungal agent in crop protection. Because of its low molecular weight and negligible structural complexity, the generation of antibodies to kresoxim-methyl noticeably requires the preparation of functionalized haptens. In this study, the introduction of a hydrocarbon spacer arm at the aromatic moieties of the target molecule was carried out by a convergent strategy based on the Sonogashira cross-coupling reaction, and functionalized linkers of the same length were also tethered to the aliphatic toxophore group by the O-alkylation reaction. Evaluation of the immune response, in ter…

Immunoanalytical approaches for the control of xenobiotics and biotoxins in food

[ES] El control efectivo de la calidad y seguridad de los ali­mentos requiere de métodos analíticos que garanticen la deter­minación fiable de cualquier sustancia potencialmente perjudicial para el consumidor que pueda estar presente en el alimento antes de su distribución y comercialización. Una de las aproximaciones analíticas que contribuye a garantizar este objetivo engloba una serie de técnicas que tienen en común la utilización de anticuerpos como elementos esenciales para la detección del analito diana, y que en conjunto reciben el nombre de métodos inmunoquímicos. Este artículo pretender aportar una visión básica de los principios bioquímicos subyacentes a estas tecnologías y cuáles…

Novel haptens and monoclonal antibodies with subnanomolar affinity for a classical analytical target, ochratoxin A

Ochratoxin A is a potent toxic fungal metabolite whose undesirable presence in food commodities constitutes a problem of public health, so it is strictly regulated and controlled. For the first time, two derivatives of ochratoxin A (OTAb and OTAd) functionalized through positions other than the native carboxyl group of the mycotoxin, have been synthesized in order to better mimic, during the immunization process, the steric and conformational properties of the target analyte. Additionally, two conventional haptens making use of that native carboxyl group for protein coupling (OTAe and OTAf) were also prepared as controls for the purpose of comparison. The immunological performance in rabbit…

Synthesis of terpenoid unsaturated 1,4-dialdehydes. Pi-facial selectivity in the Diels-Alder reaction of the 1-vinyl-2-methylcyclohexene moiety of polycyclic systems with DMAD.

Highly sensitive monoclonal antibody-based immunoassays for the analysis of fluopyram in food samples

Monoclonal antibody-based techniques have become a useful analytical technology in the agro-food sector. Nowadays, residues of the recently registered fungicide fluopyram are increasingly being found in quality control programs. In the present study, novel chemical derivatives of this pesticide were prepared and specific and high-affinity monoclonal antibodies to fluopyram were raised for the first time. Moreover, immunoassays to fluopyram were developed in two alternative enzyme-linked immunosorbent assay formats, using homologous and heterologous assay conjugates, with limits of detection below 0.05 µg L−1. The optimized immunoassays were applied to the analysis of fluopyram in fortified …

13C nuclear magnetic resonance spectra of several podocarpane and cassane diterpenoids

The 13C NMR spectra of several diterpenic derivatives having the podocarpane and cassane skeleton were recorded and interpreted. The most significant effects due to substituent orientation, B/C ring junction stereochemistry and conformational changes are briefly discussed.

New route to herbertanes via a Suzuki cross-coupling reaction: synthesis of herbertenediol

Abstract The synthesis of herbertenediol, a relevant member of the herbertane-type sesquiterpene family, is described. The synthesis is based on a new general approach to this group of sesquiterpenes where the herbertane skeleton is constructed using a Suzuki cross-coupling reaction and a [2,3]-sigmatropic Still–Wittig rearrangement as key synthetic steps.

Design and development of heterologous competitive immunoassays for the determination of boscalid residues

Boscalid is a modern agrochemical belonging to the so-called chemical class of succinate dehydrogenase inhibitor fungicides. With the aim of developing rapid analytical screening methods for this relevant compound, we herein report the synthesis of new boscalid mimics and the study of their suitability for the production of polyclonal antibodies. Aliphatic spacer arms equivalent in length and composition were tethered at two different aromatic rings of the target molecular structure. These haptens, besides being used for immunization, were employed in the development of heterologous competitive enzyme-linked immunosorbent assays (cELISAs) in order to improve assay detectability. Direct and …

Syntheses of Oxygenated Spongiane Diterpenes from Carvone. Synthesis of Dorisenone C.

Abstract The synthesis of dorisenone C, a representative member of the spongiane-type diterpene family, is described. The synthesis follows a B→AB→ABC→ABCD approach and is based on the initial preparation of the previously known hydroxy-aldehyde 14 (AB rings) from R -(−)-carvone, followed by an intramolecular Diels–Alder reaction between an oxygenated diene moiety and an acetylenic dienophile for the construction of the C ring (compound 22 ), and adequate manipulation of the Diels–Alder adduct functionality for completion of the spongiane framework.

Enantioselective synthesis of herbertane sesquiterpenes: synthesis of (−)-α-formylherbertenol

Abstract The synthesis of 4-hydroxy-3-[(1 S )-1,2,2-trimethylcyclopentyl]benzaldehyde [(−)-α-formylherbertenol 1 ], a herbertane-type sesquiterpene isolated from the leafy liverwort Herberta adunca , from β-cyclogeraniol is described. The synthesis is based on the previously described preparation of an enantiopure 1,2,2-trimethylcyclopentane synthon from which the characteristic aromatic moiety of 1 is elaborated, using a Robinson annulation and a palladium-catalysed methoxycarbonylation of an aryl triflate as key synthetic steps. The synthesis of the natural sesquiterpene (−)-α-herbertenol, also a natural sequiterpene, using the same methodology is also described.

Development of immunoaffinity columns for pyraclostrobin extraction from fruit juices and analysis by liquid chromatography with UV detection

Abstract Pyraclostrobin belongs to a new generation of fungicides widely used to preserve high valuable crops. In the present study, three monoclonal antibodies with different affinities to this modern strobilurin have been evaluated for their usefulness in the production of immunoaffinity columns suitable for the solid-phase extraction, concentration, and clean-up of residues from food commodities. Different immunosorbents were produced and characterized in terms of antibody immobilization efficiency, immunosorbent binding capacity, optimum elution conditions, and reusability. Covalent coupling of the antibodies to Sepharose–CNBr gel took place with high yield (over 90%), whereas the immun…

Immunoassays for pyraclostrobin analysis in processed food products using novel monoclonal antibodies and QuEChERS-based extracts

Strobilurins are a family of synthetic natural-derived pesticides widely used for fungi control in many crops. As part of a larger research project, a collection of monoclonal antibodies to one of the most relevant strobilurins, i.e. pyraclostrobin, has been generated using functionalized haptens with the spacer arm located at different sites. The toxophore aliphatic moiety, as observed with other strobilurins, and the planar aromatic two-ring system characteristic of this molecule were revealed as the best tethering sites for high-affinity monoclonal antibody production. A central derivatization position did not generate a good immune response, but such derivatives were useful as heterolog…

Stereoselective construction of the tetracyclic scalarane skeleton from carvone

The tetracyclic scalarane skeleton 22 has been constructed from (S)-(+)-carvone using two intramolecular Diels–Alder reactions as key synthetic steps. Abad Somovilla, Antonio, Antonio.Abad@uv.es ; Agullo Blanes, M Consuelo, Consuelo.Agullo@uv.es ; Cuñat Romero, Ana Carmen, Ana.Cunat@uv.es ; Llosa Blasco, Maria Carmen, Carmen.Llosa@uv.es

A class-selective immunoassay for simultaneous analysis of anilinopyrimidine fungicides using a rationally designed hapten

he development of multianalyte immunoassays constitutes a main research issue in the field of bioanalytical techniques. In the present study, class-specific antibodies against the three members of the anilinopyrimidine family of fungicides (pyrimethanil, cyprodinil and mepanipyrim) were raised by using a bioconjugate of a rationally designed hapten [5-(6-methyl-2-(phenylamino)pyrimidin-4-yl)pentanoic acid]. Highly sensitive immunoassays were developed for the generic determination of these compounds, using the competitive enzyme-linked immunosorbent assay (ELISA). Particularly, a direct antibody-coated competitive ELISA afforded identical sensitivity for the three anilinopyrimidines, with I…

CCDC 626412: Experimental Crystal Structure Determination

Related Article: Antonio Abad, Consuelo Agulló, Ana C. Cuñat, Ignacio de Alfonso Marzal, Antonio Gris, Ismael Navarro, Carmen Ramírez de Arellano|2007|Tetrahedron|63|1664|doi:10.1016/j.tet.2006.11.083