Fluorescence Detection of the Persistent Organic Pollutant Chlordecone in Water at Environmental Concentrations.

Chlordecone (CLD), a Persistent Organic Pollutant, is still present in water and food chain of the French West Indies (FWI), leading to dramatical public health problems. One of the major issues is the lack of an easy, non-expensive, sensitive and robust method for the detection of chlordecone to ensure chlordecone-free water and foods for the residents of the FWI. This study reports on the development of a fluorescent molecular cage that allows a simple and convenient detection of chlordecone in water at environmental concentration. The specific structural features of chlordecone prompted the choice of hemicryptophanes as receptor. First, the size, shape of the cavity, as well as the recognition units, were optimized to identify the most efficient non fluorescent host for CLD complexation. Then, this selected compound was equipped with fluorophores at different positions in order to find the most efficient system for CLD detection by fluorescence. Among the two most promising fluorescent cages, the newly synthesized hemicryptophane with biphenyl moieties allowed the development of a fast, easy, reproducible and cheap procedure to detect CLD in water. Based on its sensitivity and scalability, with modulation of hemicryptophane, concentration, CLD concentrations were estimated over five orders of magnitude (10-2 -103  µg/L) including the environmental levels of contamination and the permissible limit for drinking water in the FWI.

Elucidation of the trigonelline degradation pathway reveals previously undescribed enzymes and metabolites.

Trigonelline (TG; N-methylnicotinate) is a ubiquitous osmolyte. Although it is known that it can be degraded, the enzymes and metabolites have not been described so far. In this work, we challenged the laboratory model soil-borne, gram-negative bacterium Acinetobacter baylyi ADP1 (ADP1) for its ability to grow on TG and we identified a cluster of catabolic, transporter, and regulatory genes. We dissected the pathway to the level of enzymes and metabolites, and proceeded to in vitro reconstruction of the complete pathway by six purified proteins. The four enzymatic steps that lead from TG to methylamine and succinate are described, and the structures of previously undescribed metabolites are provided. Unlike many aromatic compounds that undergo hydroxylation prior to ring cleavage, the first step of TG catabolism proceeds through direct cleavage of the C5-C6 bound, catalyzed by a flavin-dependent, two-component oxygenase, which yields (Z)-2-((N-methylformamido)methylene)-5-hydroxy-butyrolactone (MFMB). MFMB is then oxidized into (E)-2-((N-methylformamido) methylene) succinate (MFMS), which is split up by a hydrolase into carbon dioxide, methylamine, formic acid, and succinate semialdehyde (SSA). SSA eventually fuels up the TCA by means of an SSA dehydrogenase, assisted by a Conserved Hypothetical Protein. The cluster is conserved across marine, soil, and plant-associated bacteria. This emphasizes the role of TG as a ubiquitous nutrient for which an efficient microbial catabolic toolbox is available.

De novo structure determination of 3-((3-aminopropyl)amino)-4-hydroxybenzoic acid, a novel and abundant metabolite in Acinetobacter baylyi ADP1.

INTRODUCTION: Metabolite identification remains a major bottleneck in the understanding of metabolism. Many metabolomics studies end up with unknown compounds, leaving a landscape of metabolites and metabolic pathways to be unraveled. Therefore, identifying novel compounds within a metabolome is an entry point into the 'dark side' of metabolism. OBJECTIVES: This work aimed at elucidating the structure of a novel metabolite that was first detected in the soil bacterium Acinetobacter baylyi ADP1 (ADP1). METHODS: We used high resolution multi-stage tandem mass spectrometry for characterizing the metabolite within the metabolome. We purified the molecule for 1D- and 2D-NMR (1H, 13C, 1H-1H-COSY, 1H-13C-HSQC, 1H-13C-HMBC and 1H-15N-HMBC) analyses. Synthetic standards were chemically prepared from MS and NMR data interpretation. RESULTS: We determined the de novo structure of a previously unreported metabolite: 3-((3-aminopropyl)amino)-4-hydroxybenzoic acid. The proposed structure was validated by comparison to a synthetic standard. With a concentration in the millimolar range, this compound appears as a major metabolite in ADP1, which we anticipate to participate to an unsuspected metabolic pathway. This novel metabolite was also detected in another γ-proteobacterium. CONCLUSION: Structure elucidation of this abundant and novel metabolite in ADP1 urges to decipher its biosynthetic pathway and cellular function.

Natural Chlordecone Degradation Revealed by Numerous Transformation Products Characterized in Key French West Indies Environmental Compartments.

Production and use of the insecticide chlordecone has caused long-term environmental pollution in the James River area and the French West Indies (FWI) that has resulted in acute human-health problems and a social crisis. High levels of chlordecone in FWI soils, even after its ban decades ago, and the absence of detection of transformation products (TPs), have suggested that chlordecone is virtually nonbiodegradable in the environment. Here, we investigated laboratory biodegradation, consisting of bacterial liquid cultures and microcosms inoculated with FWI soils, using a dual nontargeted GC-MS and LC-HRMS approach. In addition to previously reported, partly characterized hydrochlordecones and polychloroindenes (families A and B), we discovered 14 new chlordecone TPs, assigned to four families (B, C, D, and E). Organic synthesis and NMR analyses allowed us to achieve the complete structural elucidation of 19 TPs. Members of TP families A, B, C, and E were detected in soil, sediment, and water samples from Martinique and include 17 TPs not initially found in commercial chlordecone formulations. 2,4,5,6,7-Pentachloroindene was the most prominent TP, with levels similar to those of chlordecone. Overall, our results clearly show that chlordecone pollution extends beyond the parent chlordecone molecule and includes a considerable number of previously undetected TPs. Structural diversity of the identified TPs illustrates the complexity of chlordecone degradation in the environment and raises the possibility of extensive worldwide pollution of soil and aquatic ecosystems by chlordecone TPs.

Distinct Carbon Isotope Fractionation Signatures during Biotic and Abiotic Reductive Transformation of Chlordecone.

Chlordecone is a synthetic organochlorine pesticide, extensively used in banana plantations of the French West Indies from 1972 to 1993. Due to its environmental persistence and bioaccumulation, it has dramatic public health and socio-economic impact. Here we describe a method for carbon-directed compound specific isotope analysis (CSIA) for chlordecone and apply it to monitor biotic and abiotic reductive transformation reactions, selected on the basis of their distinct product profiles (polychloroindenes versus lower chlorinated hydrochlordecones). Significant carbon isotopic enrichments were observed for all microbially mediated transformations (εbulk = -6.8‰ with a Citrobacter strain and εbulk = -4.6‰ with a bacterial consortium) and for two abiotic transformations (εbulk = -4.1‰ with zerovalent iron and εbulk = -2.6‰ with sodium sulfide and vitamin B12). The reaction with titanium(III) citrate and vitamin B12, which shows the product profile most similar to that observed in biotic transformation, led to low carbon isotope enrichment (εbulk =-0.8‰). The CSIA protocol was also applied on representative chlordecone formulations previously used in the French West Indies, giving similar chlordecone δ13C values from -31.1 ± 0.2‰ to -34.2 ± 0.2‰ for all studied samples. This allows the in situ application of CSIA for the assessment of chlordecone persistence.

Stereochemistry and conformation of skyllamycin, a non-ribosomally synthesized peptide from Streptomyces sp. Acta 2897.

Skyllamycin is a non-ribosomally synthesized cyclic depsipeptide from Streptomyces sp. Acta 2897 that inhibits PDGF-signaling. The peptide scaffold contains an N-terminal cinnamoyl moiety, a ß-methylation of aspartic acid, three ß-hydroxylated amino acids and one rarely occurring α-hydroxy glycine. With the exception of α-hydroxy glycine, the stereochemistry of the amino acids was assigned by comparison to synthetic reference amino acids applying chiral GC-MS and Marfey-HPLC analysis. The stereochemistry of α-hydroxy glycine, which is unstable under basic and acidic conditions, was determined by conformational analysis, employing a combination of data from NOESY-NMR spectroscopy, simulated annealing and free MD simulations. The simulation procedures were applied for both R- and S-configured α-hydroxy glycine of the skyllamycin structure and compared to the NOESY data. Both methods, simulated annealing and free MD simulations independently support S-configured α-hydroxy glycine thus enabling the assignment of all stereocenters in the structure of skyllamycin and devising the role of two-component flavin dependent monooxygenase (Sky39) as S-selective.

Efficient biodegradation of the recalcitrant organochlorine pesticide chlordecone under methanogenic conditions.

Anaerobic digestion (AD) has long been studied as an effective environmental and economic strategy for treating matrices contaminated with recalcitrant pollutants. In the present work, we investigated the bioremediation potential of AD on organic waste contaminated with chlordecone (CLD), an organochlorine pesticide extensively used in the French West Indies and classified among the most persistent organic pollutants. Digestates from animal and plant origins were supplemented with CLD and incubated under methanogenic conditions for over 40 days. The redox potential and pH monitoring showed that methanogenic conditions were preserved during the entire incubation period despite the presence of CLD. In addition, the comparison of the total biogas generated from digestates with and without CLD demonstrated no adverse effects of CLD on biogas production. For the first time, a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction method, followed by GC-MS and LC-HRMS analyses, was developed to quantify CLD and its main known transformation products (TPs) in AD experiments. A decrease in CLD concentrations was evident to a greater extent under thermophilic conditions (55 °C) compared to mesophilic conditions (37.5 °C) (CLD removal of 85 % and 42 %, respectively, after 40 days of incubation). CLD degradation was confirmed by the detection and quantification of several TPs: 10-monohydroCLD (A1), two dihydroCLDs different from 2,8-dihydroCLD (A3), pentachloroindene (B1), tetrachloroindenes (B2, B3/B4), tetra- and tri-chloroindenecarboxylic acids (C1/C2, C3/C4). Determining TPs concentrations using the QuEChERS method provided an overview of CLD fate in AD. Overall, these results reveal that AD processes can efficiently degrade CLD into several TPs from A, B, and C families while maintaining satisfactory biogas production. They pave the way to developing a scaled-up AD process capable of treating CLD-contaminated organic wastes produced by farming, thus stopping any further transfer of CLD.

Is a dissipation half-life of 5 years for chlordecone in soils of the French West Indies relevant?

Recently, Comte et al. (2022) re-examined the natural degradation of chlordecone (CLD) in the soils of the French West Indies (FWI) by introducing an additional 'dissipation parameter' into the WISORCH model developed by Cabidoche et al. (2009). Recent data sets of CLD concentrations in FWI soils obtained by Comte et al. enabled them optimizing the model parameters, resulting in significantly shorter estimates of pollution persistence than in the original model. Their conclusions jeopardize the paradigm of a very limited degradation of CLD in FWI soils, which may lead to an entire revision of the management of CLD contamination. However, we believe that their study is questionable on several important aspects. This includes potential biases in the data sets and in the modeling approach. It results in an inconsistency between the estimated dissipation half-life time (DT50) of five years that the authors determined for CLD and the fate of CLD in soil from the application period 1972-1993 until nowadays. Most importantly, a rapid dissipation of CLD in the field as proposed by Comte et al. is not sufficiently supported by data and estimates. Hence, the paradigm of long-term persistence of CLD in FWI soils is still to be considered.

Access to the Syn diastereomers of cryptophane cages using HFIP.

Cryptophane cages can adopt either an anti or syn configuration that present different recognition properties. While the synthesis of anti-cryptophanes is well reported, the synthesis of syn-cryptophanes remains a challenge. Herein, we demonstrate that the use of HFIP as a co-solvent during the second ring closure reaction significantly affects the regioselectivity, providing easier access to the syn-cryptophane stereomers.

Microbiological versus Chemical Reductive Sulfidation: An Experimental and Theoretical Study.

Microbiological reductive sulfidation (RS) has rarely been documented, although it represents an efficient strategy for thiol formation. In this work, we reported on the sulfate-respiring bacterium Desulfovibrio sp.86 that has previously demonstrated RS activity toward the pesticide chlordecone. The purpose of this study was to assess its substrate versatility using a set of 28 carbonyls, to compare with chemical RS and to rationalize the observed trends using a dual experimental and theoretical approach. The chemical RS generally proceeds in two steps (S/O exchange using a sulfur donor like P4S10, reduction of the thione intermediate). Intriguingly, chlordecone was found to be converted into chlordecthiol following the first step. Hence, we designed a protocol and applied it to the 28 substrates to assess their propensity to be directly converted into thiols with the P4S10 treatment alone. Finally, we performed density functional theory calculations on these carbonyls and their thiocarbonyl derivatives to build a set of structural, electronic, and thermodynamic parameters. The results showed that chemical and microbiological RS probably involved two distinct mechanisms. Chemically, we observed that several carbonyls, possessing electron-withdrawing groups and/or aromatic rings, were directly transformed into thiols in the presence of P4S10. The correlation obtained with the electron affinity of the thiones led us to conclude that a probable single-electron reductive transfer occurred during the first step. We also found that Desulfovibrio sp.86 transformed a variety of aldehydes and ketones, without ever detecting thiones. No significant correlation was observed with the calculated parameters, but a relationship between aldehyde RS biotransformation and bacterial growth was observed. Differences in selectivity with chemical RS open the way for further applications in organic synthesis.

Microbial Transformation of Chlordecone and Two Transformation Products Formed During in situ Chemical Reduction.

Chlordecone (CLD) is a very persistent synthetic organochlorine pesticide found in the French West Indies. Recently published work has demonstrated the potential of zero-valent iron to dechlorinate CLD by in situ chemical reduction (ISCR) in soils under water-saturated conditions, forming mono- to penta-dechlorinated CLD transformation products. These transformation products are more mobile than CLD and less toxic; however, nothing is known about their further degradation, although increasing evidence of CLD biodegradation by bacteria is being found. The present study began with the enrichment from wastewater sludge of a CLD-transforming community which was then inoculated into fresh media in the presence of either CLD or two of the main ISCR transformation products, 10-monohydroCLD (-1Cl-CLD) and tri-hydroCLD (-3Cl-CLD). Carried out in triplicate batches and incubated at 38°C under anoxic conditions and in the dark, the cultures were sampled regularly during 3 months and analyzed for CLD, -1Cl-CLD, -3Cl-CLD, and possible transformation products by gas chromatography coupled to mass spectrometry. All batches showed a decrease in the amended substrates (CLD or hydroCLD). CLD degradation occurred with concomitant formation of a nine-carbon compound (pentachloroindene) and two sulfur-containing transformation products (chlordecthiol, CLD-SH; methyl chlordecsulfide, CLD-SCH3), demonstrating competing transformation pathways. In contrast, -1Cl-CLD and -3Cl-CLD only underwent a sequential reductive sulfidation/S-methylation process resulting in -1Cl-CLD-SH and -1Cl-CLD-SCH3 on the one hand, and -3Cl-CLD-SH, -3Cl-CLD-SCH3 on the other hand. Some sulfur-containing transformation products have been reported previously with single bacterial strains, but never in the presence of a complex microbial community. At the end of the experiment, bacterial and archaeal populations were investigated by 16S rRNA gene amplicon sequencing. The observed diversity was mostly similar in the CLD and -1Cl-CLD conditions to the inoculum with a dominant archaea genus, Methanobacterium, and four OTU affiliated to bacteria, identified at the family (Spirochaetaceae) or genus level (Desulfovibrio, Aminobacterium, and Soehngenia). On the other hand, in the -3Cl-CLD condition, although the same OTU were found, Clostridium sensu stricto 7, Candidatus Cloacimonas, and Proteiniphilum were also present at > 2% sequences. Presence of methanogens and sulfate-reducing bacteria could contribute to sulfidation and S-methylation biotransformations. Overall, these results contribute to increasing our knowledge on the biodegradability of CLD and its transformation products, helping to progress toward effective remediation solutions.

Transformation of the recalcitrant pesticide chlordecone by Desulfovibrio sp.86 with a switch from ring-opening dechlorination to reductive sulfidation activity.

The insecticide chlordecone has been used in the French West Indies for decades, resulting in long term pollution, human health problems and social crisis. In addition to bacterial consortia and Citrobacter sp.86 previously described to transform chlordecone into three families of transformation products (A: hydrochlordecones, B: polychloroindenes and C: polychloroindenecarboxylic acids), another bacterium Desulfovibrio sp.86, showing the same abilities has been isolated and its genome was sequenced. Ring-opening dechlorination, leading to A, B and C families, was observed as previously described. Changing operating conditions in the presence of chlordecone gave rise to the formation of an unknown sulfur-containing transformation product instead of the aforementioned ones. Its structural elucidation enabled to conclude to a thiol derivative, which corresponds to an undocumented bacterial reductive sulfidation. Microbial experiments pointed out that the chlordecone thiol derivative was observed in anaerobiosis, and required the presence of an electron acceptor containing sulfur or hydrogen sulfide, in a confined atmosphere. It seems that this new reaction is also active on hydrochlordecones, as the 10-monohydrochlordecone A1 was transformed the same way. Moreover, the chlordecone thiol derivative called F1 was detected in several chlordecone contaminated mangrove bed sediments from Martinique Island, highlighting the environmental relevance of these results.

Genetic Analysis of Citrobacter sp.86 Reveals Involvement of Corrinoids in Chlordecone and Lindane Biotransformations.

Chlordecone (Kepone®) and γ-hexachlorocyclohexane (γ-HCH or lindane) have been used for decades in the French West Indies (FWI) resulting in long-term soil and water pollution. In a previous work, we have identified a new Citrobacter species (sp.86) that is able to transform chlordecone into numerous products under anaerobic conditions. No homologs to known reductive dehalogenases or other candidate genes were found in the genome sequence of Citrobacter sp.86. However, a complete anaerobic pathway for cobalamin biosynthesis was identified. In this study, we investigated whether cobalamin or intermediates of cobalamin biosynthesis was required for chlordecone microbiological transformation. For this purpose, we constructed a set of four Citrobacter sp.86 mutant strains defective in several genes belonging to the anaerobic cobalamin biosynthesis pathway. We monitored chlordecone and its transformation products (TPs) during long-term incubation in liquid cultures under anaerobic conditions. Chlordecone TPs were detected in the case of cobalamin-producing Citrobacter sp.86 wild-type strain but also in the case of mutants able to produce corrinoids devoid of lower ligand. In contrast, mutants unable to insert the cobalt atom in precorrin-2 did not induce any transformation of chlordecone. In addition, it was found that lindane, previously shown to be anaerobically transformed by Citrobacter freundii without evidence of a mechanism, was also degraded in the presence of the wild-type strain of Citrobacter sp.86. The lindane degradation abilities of the various Citrobacter sp.86 mutant strains paralleled chlordecone transformation. The present study shows the involvement of cobalt-containing corrinoids in the microbial degradation of chlorinated compounds with different chemical structures. Their increased production in contaminated environments could accelerate the decontamination processes.

3,5,7-Tripropyl-1-aza-adamantane-4,6,10-triol.

The title compound, C(18)H(33)NO(3), was prepared according to a highly diastereoselective hydrogenation procedure from 3,5,7-triallyl-1-aza-adamantane-4,6,10-trione. The crystal structure of the title compound contains two crystallographically independent mol-ecules (Z' = 2), which are linked by inter-molecular hydrogen bonding into chains. In contrast to the aza-adamantanones, the aza-adamantanetriol core of the title compound does not show any particular C-C bond elongation.

Microbial Degradation of a Recalcitrant Pesticide: Chlordecone.

Chlordecone (Kepone®) is a synthetic organochlorine insecticide (C10Cl10O) used worldwide mostly during the 1970 and 1980s. Its intensive application in the French West Indies to control the banana black weevil Cosmopolites sordidus led to a massive environmental pollution. Persistence of chlordecone in soils and water for numerous decades even centuries causes global public health and socio-economic concerns. In order to investigate the biodegradability of chlordecone, microbial enrichment cultures from soils contaminated by chlordecone or other organochlorines and from sludge of a wastewater treatment plant have been conducted. Different experimental procedures including original microcosms were carried out anaerobically over long periods of time. GC-MS monitoring resulted in the detection of chlorinated derivatives in several cultures, consistent with chlordecone biotransformation. More interestingly, disappearance of chlordecone (50 µg/mL) in two bacterial consortia was concomitant with the accumulation of a major metabolite of formula C9Cl5H3 (named B1) as well as two minor metabolites C10Cl9HO (named A1) and C9Cl4H4 (named B3). Finally, we report the isolation and the complete genomic sequences of two new Citrobacter isolates, closely related to Citrobacter amalonaticus, and that were capable of reproducing chlordecone transformation. Further characterization of these Citrobacter strains should yield deeper insights into the mechanisms involved in this transformation process.

Trimerization products of trifluoroacetone: critical solvent effect on position and kinetics of anomeric equilibria.

In the presence of bases, trifluoroacetone is known to trimerize leading to configurationally labile 6-methyl-2,4,6-tris(trifluoromethyl)tetrahydro-2H-pyran-2,4-diols 1a,b and 2a,b, structurally close to fluorinated carbohydrates. We report herein a complete study of their behavior in solution. The remarkable solvent effect on the two equilibria (1a <--> 1b; 2a <--> 2b) was rationalized using solvent basicity measures and polarity scales. Solvents of weak donor number were found to favor the diastereoisomers 1a and 2a, which were subsequently isolated. According to their X-ray analyses, they both possess a concave structure with 1,3-cis-diaxial hydroxyl groups. A complementary kinetic study illustrated that acidic conditions can drastically reduce the equilibration rate, allowing the use of a wide range of solvents. Finally, a reexamination of previously published trimerization conditions using sodium or magnesium amalgam revealed that, contrary to the suggestion by two independent reports, 1,3,5-tris(trifluoromethyl)cyclohexane-1,3,5-triol 3/4 was neither formed as the principal product in place of 1a,b and 2a,b nor could it be detected as a minor product.

Nucleophilic substitution by grignard reagents on sulfur mustards.

With proper activation of the leaving group, sulfur mustards react with Grignard reagents with neighboring group participation of the sulfur atom. 2,6-Dichloro-9-thiabicyclo[3.3.1]nonane is especially useful in this regard, providing clean reactivity with organomagnesium nucleophiles on a topologically constrained scaffold.