Quinolizidine alkaloids in lupine flour and lupine products from the German retail market and risk assessment of the results regarding human health.

This study describes the analysis of five quinolizidine alkaloids (QA), i.e. 13-OH-lupanine, lupanine, lupinine, angustifoline and sparteine in 30 samples of lupine flours, lupine seeds and derived products collected 2019-2021 on the German retail market. QA occur as secondary metabolites in plants of lupine species. Certain QA are of toxicological relevance. The analytical determination performed by LC-MS/MS revealed some samples with high QA concentrations (up to 21,000 mg/kg), particularly in bitter lupine seeds. As those concentrations would result in substantial exceedances of the maximum tolerable intake values proposed by health authorities, they must be considered as a health concern.

Determination of pyrrolizidine alkaloids in tea and honey with automated SPE clean-up and ultra-performance liquid chromatography/tandem mass spectrometry.

For routine analytical purpose a method based on a combination of automated solid-phase extraction (SPE) clean-up and detection by Ultra-Performance Liquid Chromatography/Tandem Mass Spectrometry (UPLC-MS/MS) was developed for the determination of pyrrolizidine alkaloids (PA) in various food commodities. In this survey, honey, tea and herbal infusion samples from local retailers collected in 2012-2015 were obtained and analysed for their PA content. PA concentrations were found in 30% of the honey samples and in 42% of the tea and herbal infusion samples with levels up to 595 µg/kg. The survey included 17 individual PA, and their sum is also reported for each sample.

Evidence of ochratoxin A conjugates in urine samples from infants and adults.

Ochratoxin A (OTA), a mycotoxin with nephrotoxic and carcinogenic properties, is an important contaminant of food and feed. Analysis of OTA in human biological fluids (blood, urine, or breast milk) has documented frequent exposure to this mycotoxin, yet at quite variable levels in different population groups across the world. Urine is the preferred matrix in biomonitoring since sample collection is non-invasive and better accepted by study participants. As only a small fraction of the ingested OTA is excreted in urine, determination of urinary OTA requires sensitive analytical techniques, and phase-II-metabolites should be also considered as biomarkers of exposure. Yet, data published so far on the presence of OTA-glucuronide/sulfate in human urine have been contradictory. In this study, urines (n = 38) from two groups of breastfed infants (German and Turkish) and from German adults were now analysed for the presence of OTA glucuronides or sulfates by an indirect method, i.e. by comparing the levels of OTA (aglycone) in urines without and after enzymatic hydrolysis with ß-glucuronidase/arylsulfatase. Additionally, ochratoxin A-8-ß-glucuronide and open lactone ochratoxin A-8-ß-glucuronide were synthesized to serve as reference materials for metabolite analysis. Attempts for definitive confirmation of glucuronides of OTA via direct identification in LC-MS/MS analysis were hampered by the lower ionizability of the conjugates compared to the parent compound. Considerable increases in OTA levels were found after enzymatic hydrolysis in several (not all) urine samples and provide clear evidence for the excretion of OTA-conjugates. The latter observation is of importance, since OTA phase-II-metabolites may escape detection when direct methods are applied for urinary biomarker analysis. In conclusion, enzymatic hydrolysis of urine samples is highly advisable in order to avoid an underestimation of the OTA-exposure.

Two histone deacetylases, FfHda1 and FfHda2, are important for Fusarium fujikuroi secondary metabolism and virulence.

Histone modifications are crucial for the regulation of secondary metabolism in various filamentous fungi. Here we studied the involvement of histone deacetylases (HDACs) in secondary metabolism in the phytopathogenic fungus Fusarium fujikuroi, a known producer of several secondary metabolites, including phytohormones, pigments, and mycotoxins. Deletion of three Zn(2+)-dependent HDAC-encoding genes, ffhda1, ffhda2, and ffhda4, indicated that FfHda1 and FfHda2 regulate secondary metabolism, whereas FfHda4 is involved in developmental processes but is dispensable for secondary-metabolite production in F. fujikuroi. Single deletions of ffhda1 and ffhda2 resulted not only in an increase or decrease but also in derepression of metabolite biosynthesis under normally repressing conditions. Moreover, double deletion of both the ffhda1 and ffhda2 genes showed additive but also distinct phenotypes with regard to secondary-metabolite biosynthesis, and both genes are required for gibberellic acid (GA)-induced bakanae disease on the preferred host plant rice, as Δffhda1 Δffhda2 mutants resemble the uninfected control plant. Microarray analysis with a Δffhda1 mutant that has lost the major HDAC revealed differential expression of secondary-metabolite gene clusters, which was subsequently verified by a combination of chemical and biological approaches. These results indicate that HDACs are involved not only in gene silencing but also in the activation of some genes. Chromatin immunoprecipitation with the Δffhda1 mutant revealed significant alterations in the acetylation state of secondary-metabolite gene clusters compared to the wild type, thereby providing insights into the regulatory mechanism at the chromatin level. Altogether, manipulation of HDAC-encoding genes constitutes a powerful tool to control secondary metabolism in filamentous fungi.

Segregation of secondary metabolite biosynthesis in hybrids of Fusarium fujikuroi and Fusarium proliferatum.

Fusarium fujikuroi and Fusarium proliferatum are two phylogenetically closely related species of the Gibberella fujikuroi species complex (GFC). In some cases, strains of these species can cross and produce a few ascospores. In this study, we analyzed 26 single ascospore isolates of an interspecific cross between F. fujikuroi C1995 and F. proliferatum D4854 for their ability to produce four secondary metabolites: gibberellins (GAs), the mycotoxins fusarin C and fumonisin B(1), and a family of red polyketides, the fusarubins. Both parental strains contain the biosynthetic genes for all four metabolites, but differ in their ability to produce these metabolites under certain conditions. F. fujikuroi C1995 produces GAs and fusarins, while F. proliferatum D4854 produces fumonisins and fusarubins. The segregation amongst the progeny of these traits is not the expected 1:1 Mendelian ratio. Only eight, six, three and three progeny, respectively, produce GAs, fusarins, fumonisin B(1) and fusarubins in amounts similar to those synthesized by the producing parental strain. Beside the eight highly GA(3)-producing progeny, some of the progeny produce small amounts of GAs, predominantly GA(1), although these strains contain the GA gene cluster of the non-GA-producing F. proliferatum parental strain. Some progeny had recombinant secondary metabolite profiles under the conditions examined indicating that interspecific crosses can yield secondary metabolite production profiles that are atypical of the parent species.

Effects of oral exposure of pigs to deoxynivalenol (DON) sulfonate (DONS) as the non-toxic derivative of DON on tissue residues of DON and de-epoxy-DON and on DONS blood levels.

The Fusarium toxin deoxynivalenol (DON) is of outstanding importance in pig nutrition because of its frequent occurrence in cereal grains at levels high enough to cause adverse effects such as a decrease in feed intake and impairment of the immune system. Thus, simple decontamination procedures would be useful. The present study aimed to examine the effects of wet preservation of triticale contaminated with DON and zearalenone (ZON) with sodium metabisulphite (SBS) on the treatment-related non-toxic derivative of DON (DON-sulfonate, DONS), and on ZON and its metabolites in blood and various physiological specimens of piglets. The uncontaminated control triticale (CON) and the DON-contaminated triticale (FUS) were included in the diets either untreated or SBS treated (CON-SBS, FUS-SBS) and fed to piglets for 28 days starting from weaning. The diet concentrations for DON were 0.156, 0.084, 2.312 and 0.275 mg kg(-1), for DONS were <0.05, <0.05, <0.05 and 1.841 mg kg(-1), and for ZON were <0.001, 0.006, 0.017, and 0.016 mg kg(-1) for each of CON, CON-SBS, FUS and FUS-SBS, respectively. DONS was present in the blood of piglets fed the FUS-SBS at a median concentration of 15.5 ng ml(-1) (3-67 ng ml(-1)), while the median DON concentration amounted to 2 ng ml(-1) (0-5 ng ml(-1)) at the same time. The median DON concentration in the blood of piglets fed the FUS diet reached a median concentration of 10.5 ng ml(-1) (5-17 ng ml(-1)). Moreover, the relative differences between the DON concentrations in other physiological specimens (muscle, liver, kidney, bile and urine) in piglets fed the FUS-SBS and the FUS diet were comparable with the blood DON concentration differences. Although these differences can be taken as an indication for DONS stability after absorption and distribution further studies examining DONS in these other physiological specimens directly are necessary to substantiate this conclusion. Moreover, ZON and α-zearalenol could only be detected in bile and urine where their levels were not influenced by the SBS treatment.

Effects of the mycotoxin fumonisin B(1) on cell death in human kidney cells and human lung fibroblasts in primary culture.

Fumonisins are mycotoxins produced by Fusarium verticillioides. The toxic effects of fumonisin B(1) (FB(1)) at the cellular level consist of a mixture of both necrosis and apoptosis. We studied the effect of FB(1) in human lung fibroblasts (NHLF) and human kidney epithelial cells (RPTEC) in primary culture. Apoptotic and necrotic cell death, collagen and fibronectin secretion were determined mainly after 14 days' exposure. The protein content of NHLF and RPTEC cells was slightly increased after 14 days' exposure to low FB(1) concentrations (0.1 or 1 microm). Caspase-3 activity tended to increase in NHLF and to decrease in RPTEC cells with higher FB(1) concentrations after 14 days' exposure. LDH release was slightly decreased in both cell types after 14 days. Collagen I and III secretion was enhanced in NHLF cells. Collagen III was decreased in RPTEC. Collagen IV was not changed in both cell types. Fibronectin secretion was uninfluenced in RPTEC and interim increased in NHLF. Furthermore LC-MS/MS studies did not give any hints for a metabolism of FB(1). Therefore, the main risk of prolonged FB(1) exposure seems to be altered collagen secretion pattern.

[Spargelstangenuntersuchungen zur Haupterntezeit auf Infektionen mitFusarium spp. und Kontaminationen mit Fumonisin B1 Investigation on asparagus spears during the main harvest byFusarium spp.- infections and contamination by Fumonisin B1].

Asparagus spears collected from a total of six commercial plantings in Austria during the main harvest periods in May and June of 2003 and 2004 were examined for endophytic colonization byFusarium spp., particularlyF. proliferatum. Potentially toxigenic fungi such asF. proliferatum were isolated and identified by morphological characteristics using light microscopy. Fumonisin B1 inF. proliferatum-infected asparagus spears was detected with IAS-HPLC-FLD or HPLC-MS/MS. The identity of endophytic fungi colonizing of a total of 816 individual spears was determined. The incidence of infection byF. proliferatum and otherFusarium spp. was highly dependent on location and sampling date. The dominantFusarium species among the endophytic microflora wasF. oxysporum. Other frequently isolated species includedF. proliferatum, F. sambucinum, F. culmorum, F. avenaceum andF. equiseti. The incidence ofF. proliferatum-infected asparagus spears was less than 10% at four of the six sampling locations. At the two remaining locations, 20-47% of the spears examined were infected withF. proliferatum. Further exploration of FB1 generation in asparagus is required because the low levels of FB1 (10-50 (µg/kg) detected in harvested spears in 2003 and 2004 cannot be explained by the results of this study.

[Isolation and structural elucidation of thermal degradation products of theFusarium mycotoxin nivalenol]. / Isolierung und Strukturaufklärung von thermischen Abbauprodukten des Mykotoxins Nivalenol.

The major class of mycotoxins produced byFusarium moulds are trichothecenes, a large group of sesquiterpenes sharing the same basic chemical structure, a 12,13-epoxytrichothec-9-ene ring system. Their toxicity is attributed to their ability to noncompetitively inhibit the biosynthesis of proteins in eukaryotic cells. Trichothecenes in general are relatively stable substances and their degradation is reported only at high temperatures and prolonged heating time.In an attempt to investigate the stability of the trichothecene nivalenol (NIV) under food processing conditions such as cooking or baking, we performed a number of experiments using a model heating system.Heating of nivalenol, especially under mild alkaline conditions, gave a mixture of four compounds (norNIV A, norNIV B, norNIV C and NIV lactone), which were separated and further analyzed by gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS). Structure elucidation was achieved by 1D and 2D nuclear magnetic resonance (NMR) experiments.We further demonstrated the formation of these products in heating experiments with spiked flour samples. In a screening of several commercially available products only norNIV B was detected in one of the samples, possibly due to the very low contamination of these particular samples with nivalenol.

Combinatory effects of citrinin and ochratoxin A in immortalized human proximal tubule cells.

Ochratoxin A (OTA) and citrinin (CIT) are two mycotoxins often occurring together in grains and cereals. Although both are nephrotoxic and can induce apoptosis, combination effects have not been examined up to now. Therefore, the aim of this study was to take a close look at the interactions of citrinin and OTA in cultured human proximal tubule-derived cells (IHKE cells). The cytotoxicity of both mycotoxins was studied, measuring the metabolic activity and the cell number. Furthermore, caspase 3-activation as a marker for apoptosis was examined for both mycotoxin alone and in combination. The results show that citrinin had an antagonistic effect on ochratoxin A induced caspase 3-activation in concentrations of 2.5 and 5 µmol/l. Higher concentrations (7.5 and 15 µmol/l) lead to additive effects, lower citrinin concentrations (0.25 and 1 µmol/l) did not show any effect at all. The observed decrease in caspase 3-activity was specific for the combination with OTA, since the combination of citrinin with cisplatin did not show any effect. Citrinin did not influence of the OTA-induced apoptosis when added two hours after applying ochratoxin A. Also the combination of both toxins decreased the uptake of OTA into the cells which might be an explanation for the antagonistic effect of citrinin in certain concentrations. However, the transport into cells can not be the only explanation. so further examinations are necessary.

Quantitative analysis of Fusarium mycotoxins in maize using accelerated solvent extraction before liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry.

A method for the simultaneous quantitative determination of deoxynivalenol (DON), fumonisin B1 (FB1) and zearalenone (ZEN) in maize by liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCIMS/MS), using stable isotopically labelled and structural analogues internal standards, is described. The procedure involves accelerated solvent extraction followed by two solid-phase clean-up steps on strong anion exchange resin and a Mycosep column. Typical recoveries were calculated by spiking blank maize at three different concentrations for deoxynivalenol (200, 400 and 1000 microg kg(-1)) at 70%, for fumonisin B1 (100, 200 and 1000 microg kg(-1)) at 90%, and for zearalenone (50, 100 and 200 microg kg(-1)) at 40%. LC-APCIMS/MS analyses were realized in collision-induced dissociation on an ion-trap instrument to provide a high degree of selectivity and sensitivity. Extraction of ions from two transition reactions, monitored by LC-APCIMS/MS for each analyte, enabled a limit of detection for DON, FB1 and ZEN at, respectively, 10, 20 and 3 microg kg(-1), and a limit of quantification at, respectively, 50, 50 and 10 microg kg(-1). The robustness of the method was also evaluated with the analysis of wheat samples.

Occurrence of fumonisins in asparagus (asparagus officinalis L.) and garlic (allium sativum L.) from Germany.

Fusarium proliferatum is able to produce fumonisins and is considered a pathogen of many economically important plants (e.g. corn, rice, asparagus) [1]. The occurrence of fumonisin FB1 inF. proliferatum infected asparagus spears from Germany was investigated using a liquid chromatography/electrospray ionization-mass spectrometry (LC-ESI-MS) method with isotopically labeled fumonisin FB1-d6 as internal standard. Asparagus samples were harvested in July 2000 and screened forFusarium species. AltogetherF. oxysporum, F. proliferatum and F. sambucinum were isolated from the spears. The samples infected with F.proliferatum were subsequently analyzed for fumonisins. FB1 was detected in 9 of the 10 samples in amounts ranging from 36.4 ng/g to 4513.7 ng/g (based on dry weight). Fumonisins FB2 and FB3 were found in six samples in lower concentrations. In asparagus spears of June 2002 we could findF. proliferatum in 6% of the samples, however no fumonisins were detectable.Furthermore the capability of producing FB1 by the fungus in garlic bulbs was investigated. Therefore garlic was cultured inF. proliferatum contaminated soil and the bulbs were screened for infection with F.proliferatum and for the occurrence of fumonisins by LC-MS. F.proliferatum was detectable in the garlic tissue and all samples contained FB1 (26.0 ng/g to 94.6 ng/g).This is the first report of the natural occurrence of FB1 in German asparagus spears and furthermore our findings suggest a potential for natural contamination of garlic bulbs with fumonisins. For detailed results and methods see Ref. [2].

Induction of apoptosis in cultured human proximal tubule cells by fumonisins and fumonisin metabolites.

Fumonisin B1 (FB1) causes apoptosis in a variety of cell types and tissues but the apoptotic potential of other fumonisins and fumonisin metabolites has not been determined and the underlying mechanisms are not completely understood. In our studies we exposed human proximal tubule-derived cells (IHKE cells) to FB1, fumonisin B2 (FB2), fumonisin B3 (FB3), hydrolyzed fumonisin B1 (HFB1) and N-palmitoyl-hydrolyzed fumonisin B1 (N-Pal-HFB1) and investigated caspase-3 activation, chromatin condensation and DNA fragmentation. Exposure to 10 micromol/L FB1 for 24 h led to a significant increase in caspase-3 activity, chromatin condensation and to DNA fragmentation. All other tested compounds did not show any significant activation of caspase-3 activity nor chromatin condensation and DNA-fragmentation. Furthermore, we examined if a sphinganine accumulation is correlated with an induction of apoptosis in IHKE cells. Therefore we used a liquid chromatography/electrospray ionization-mass spectrometry(LC/ESI-MS)-method using phytosphingosine as an internal standard to determine sphinganine and sphingosine concentrations in IHKE cells. Whereas a significant increase of sphinganine (up to 7000% compared to control cells) was observed with all fumonisin-derivates, sphingosine levels nearly remained unchanged indicating that all substrates inhibited ceramide synthase effectively. These results demonstrate that all compounds let to increased sphinganine levels in IHKE cells but only FB1 was able to induce apoptosis. We conclude that the inhibition of the ceramide synthase is not per se a predictor whether or not fumonisins induce apoptosis.

Liquid chromatography/electrospray ionisation-mass spectrometry method for the quantification of sphingosine and sphinganine in cell cultures exposed to fumonisins.

Fumonisins, mycotoxins produced by Fusarium verticillioides, are potent inhibitors of the de novo sphingolipid biosynthesis via inhibition of the key enzyme ceramide synthase. The cellular response to a fumonisin exposure is obvious as an alteration of the ratio of the sphingoid bases sphingosine (SO) and sphinganine (SA). We developed a new column liquid chromatography/electrospray ionisation-mass spectrometry (LC-ESI-MS) method for the rapid, simultaneous and quantitative determination of these bases in cell cultures of immortalised human kidney epithelial cells (IHKE cells). For sample preparation, cell lysates were only diluted, centrifuged and directly used for LC-MS measurements. Quantification was carried out using phytosphingosine (PSO) as an internal standard. Detecting the protonated molecule [M+H](+) signals of SO (m/z 300) and SA (m/z 302) in the selected ion monitoring (SIM) mode, detection limits of 10 pg for SO (signal-to-noise ratio S/N=3:1) and 25 pg for SA (S/N=3:1) were established. The average recovery for SO and SA was higher than 90% for control IHKE-cells, respectively. The developed LC-ESI-MS method allows the sensitive, selective and rapid monitoring of sphingosine and sphinganine in cell matrices with a drastically reduced time for sample preparation.

Formation of fumonisin artefacts in thermal treated food.

In order to study the formation of fumonisin artefacts and the binding of fumonisins to matrix components (e.g. starch and protein) in thermal treated food, model experiments were performed by heating fumonisin FB1 with amino acid derivatives (protein model) and methyl-a-D-glucopyranoside (starch model). The reaction products were analysed by highperformance liquid chromatography-electrospray ionisationtandem mass spectrometry (HPLC-MS/MS). Using MS/MS experiments the formed reaction products were characterized as conjugates between fumonisin B1 and the used substrates. The reaction product between fumonisin B1 and methyl-α-D-glucopyranoside was purified and identified by nuclear magnetic resonance (NMR) spectroscopy as the diester of fumonisin B1 and methyl-α-D-glucopyranoside. These studies indicate that fumonisins can bind to matrix components via their TCA side chains.

Analyses of red fermented rice (angkak) and report of a newMonascus metabolite.

A HPLC-based method for the analysis of red fermented rice and results obtained with it are presented. Formation of citrinin, red, orange and yellow pigments byMonascus depends on the culture substrate. Citrinin and some pigments are decomposed by heat. A newMonascus metabolite, monascodilone, its structure and preliminary data on its toxicity are reported.

A new one-step strategy for the stereochemical assignment of acyclic 2- and 3-sulfanyl-1-alkanols using the CD exciton chirality method.

A new one-step strategy is described for the stereochemical assignment of acyclic 2- and 3-sulfanyl-1-alkanols using the CD exciton chirality method. Using the 9-anthroate chromophore for the derivatization of both functional groups, the resulting bisignate CD curves unequivocally allow the determination of the stereochemistry from a single CD measurement. The usefulness of the new method is demonstrated using synthesized optically pure 3-sulfanyl-1-hexanols and 2-sulfanyl-1-hexanols as model compounds. The developed microscale method is also useful for the stereochemical assignment of 1,2- and 1,3-diols. To our knowledge this is the first application of the CD exciton chirality method to acyclic 2- and 3-sulfanyl-1-alkanols.

Enantioselective epoxidation with chiral MN(III)(salen) catalysts: kinetic resolution of aryl-substituted allylic alcohols.

A set of aryl-substituted allylic alcohols rac-2 has been epoxidized by chiral Mn(salen*) complexes 1 as the catalyst and iodosyl benzene (PhIO) as the oxygen source. Whereas one enantiomer of the allylic alcohol 2 is preferentially epoxidized to give the threo- or cis-epoxy alcohol 3 (up to 80% ee) as the main product (dr up to >95:5), the other enantiomer of 2 is enriched (up to 53% ee). In the case of 1,1-dimethyl-1,2-dihydronaphthalen-2-ol (2c), the CH oxidation to the enone 4c proceeds enantioselectively and competes with the epoxidation. The absolute configurations of the allylic alcohols 2 and their epoxides 3 have been determined by chemical correlation or CD spectroscopy. The observed diastereo- and enantioselectivities in the epoxidation reactions are rationalized in terms of a beneficial interplay between the hydroxy-directing effect and the attack along the Katsuki trajectory.

Determination of N-(carboxymethyl)fumonisin B(1) in corn products by liquid chromatography/electrospray ionization--mass spectrometry.

It is well-known that fumonisin B(1) (FB(1)) in corn meal decreases during baking, frying, and cooking, but it is still not exactly clear how heating affects the formation of N-(carboxymethyl)fumonisin B(1) (NCM-FB(1)), the reaction product of FB(1) and reducing sugars. In model experiments corn grits were spiked with FB(1) (2 mg/kg) and D-glucose (50 g/kg) or sucrose (50 g/kg) and manufactured into extrusion products at various temperatures (160--180 degrees C) and moisture levels (16--20%). A liquid chromatography/electrospray ionization-mass spectrometry method using isotopically labeled fumonisin FB(1)-d(6) as an internal standard was developed for the determination of NCM-FB(1). For sample cleanup solid-phase C18 cartridges were used. The detection limit achieved with this method was 10 ng/g (signal-noise ratio = 3:1) using the protonated molecule [M + H](+) signal of NCM-FB(1) (m/z 780) in the selected ion monitoring mode. Low concentrations of NCM-FB(1) (29-97 ng/g) were detected in all samples spiked with D-glucose and FB(1), whereas those spiked with FB(1) and sucrose showed only NCM-FB(1) in samples produced at 180 degrees C (NCM-FB(1) = 27 ng/g). Various corn-containing food samples from the German market were analyzed for the presence of NCM-FB(1), FB(1), and hydrolyzed fumonisin B(1) (HFB(1)). All samples were contaminated with FB(1) (22--194 ng/g) and HFB(1) (5--247 ng/g). Six of nine samples contained NCM-FB(1) in low concentrations ranging from 10 to 76 ng/g. From these data and the low toxicity of NCM-FB(1) it can be concluded that the significance of NCM-FB(1) in food seems to be a minor one.

Combined synthetic/CD strategy for the stereochemical assignment of the tricarballylic acid side chains of fumonisin B(1).

The circular dichroism (CD) exciton chirality method was employed for the stereochemical assignment of the tricarballylic acid (TCA) side chains of fumonisin B(1) 1a (FB(1)). Using 2-naphthoate for chromophoric derivatization of the reduced TCA moieties, the absolute configuration was shown to be R. For additional confirmation, an optically active dihydroxy-tert-butanoate 2 related to the TCA group of fumonisin B(1) was synthesized to serve as a model compound.