Rates of Oxidative Coupling of Humic Phenolic Monomers Catalyzed by a Biomimetic Iron-Porphyrin

A synthetic water-soluble meso-tetra(2,6-dichloro-3-sulfonatophenyl)porphyrinate of iron(III) chloride (FeP) was used as biomimetic catalyst in the oxidative coupling of three monomeric phenols (catechol, caffeic, and p-coumaric acids), which are common constituents of natural humic substances. The extent of oxidation induced by the FeP catalyst in solutions of phenolic monomers was followed in the presence of an oxygen donor such as hydrogen peroxide or dissolved oxygen under daylight radiation. Both UV- and fluorescence-detected liquid chromatograms indicated that primary oxidation products had a larger electronic conjugation and molecular mass than the original phenols, thereby suggesting that the biomimetic oxidative catalysis produced covalently linked phenylene and oxyphenylene oligomers. However, the polyphenolic products were further oxidized in the progress of the catalytic reaction to possible undetectable aliphatic acids or even to complete mineralization. Rate constants describing the initial reaction period were larger for the catalyzed oxidation with hydrogen peroxide than those for the noncatalyzed control solutions under autoxidation or hydrogen peroxide treatment. However, the rate constants measured for the phenol solutions treated with just the FeP catalyst showed that the presence of dissolved oxygen and the action of the daylight radiation were sufficient to significantly increase the reaction rate in respect to control solutions. These results confirmed previous findings, showing that humic materials may undergo oxidative coupling catalyzed by metal-porphyrins in the presence of either an oxygen donor or, simply, dissolved molecular oxygen under daylight. The increase of molecular mass of natural humic and polyphenolic substances by this biomimetic technology may have useful applications in environmental chemistry

Molecular Rigidity and Diffusivity of Al³⁺ And Ca²⁺ Humates As Revealed by NMR Spectroscopy

NMR techniques were applied to follow changes in molecular rigidity and diffusion of complexes formed between a humic acid (HA) and either aluminum or calcium ions, added in amounts ranging from 0.05% to 1% of HA carboxylic acidity. Spin−lattice relaxation time in the rotating frame (T1ρ(H)) and diffusion coefficients (D) of humic−metal complexes were obtained from 13C cross-polarization magic angle spinning (CP-MAS), and 1H-diffusion order spectroscopy (DOSY) spectra, respectively. Molecular rigidity of humic complexes increased significantly with metal addition throughout the full carbon spectral region, being more pronounced for triple-charged Al than for double-charged Ca. However, T1ρ(H) values of spectral intervals suggested that molecular rigidity increase was generally in the following order: aliphatic C > aromatic/double bonds C > carboxyl C. Concomitantly, DOSY spectra showed that addition of both Al and Ca decreased substantially the diffusivity of humic alkyl components and increased that of aromatic and hydroxyalkyl components, thereby indicating that complexation induced a molecular-size increase in the former and a decrease in the latter. These results suggest that saturated and unsaturated long-chain alkanoic acids in HA were preferentially involved in metal complexation with Al and Ca, with consequent increase of conformational rigidity and molecular size of humic hydrophobic domains. Conversely, more hydrophilic or mobile humic components appeared relatively less affected by the molecular and intermolecular rearrangements induced in HA by complexation with metals. Such NMR approach appears thus liable to evaluate the response to metal complexation of specific chemical entities present in the bulk HA and provides a further insight in the molecular architecture of humic−metal complexes

Host-Guest Interactions between 2,4-Dichlorophenol and Humic Substances As Evaluated by ¹H NMR Relaxation and Diffusion Ordered Spectroscopy

1H NMR measurements of spin−lattice (T1) and spin−spin (T2) relaxation times and diffusion ordered spectroscopy (DOSY) were applied to investigate the association of 2,4-dichlorophenol (DCP) with a soil fulvic (FA−VICO) and humic acid (HA−-VICO), and a lignite humic acid (HA−LIG). The 1H T1 and T2 values of DCP were found to decrease with increasing humic concentration, indicating reduction in molecular mobility due to formation of noncovalent interactions. The increased shortening of relaxation times observed upon addition of HA suggested more extensive association of DCP with HA than with FA. The extent of binding was inferred from diffusion coefficients (D) which showed slower diffusion for bound DCP. At 1 mg mL−1 DCP was completely bound by 4.1 and 5.8 mg mL−1 of HA−VICO and HA−LIG, respectively, while full DCP association was not observed even up to 20 mg mL−1 of FA. This was reflected by association constants (Ka): 3.1 ± 0.3 M−1 for FA−DCP, and 15.5 ± 3.1 M−1 and 11.0 ± 1.2 M−1 for HA−VICO and HA−LIG DCP complexes, respectively. The stronger binding to HA is attributed to their larger hydrophobic character enabling formation of stable hydrophobic domains to which DCP becomes associated in host−guest complexes. DCP complexation within humic hydrophobic domains was confirmed by upfield chemical shifts and signal line broadenings observed in 1H NMR spectra. Similar chemical shift variations for the three DCP aromatic protons further indicated π−π interactions, rather than H-bonding, as the main driving force for noncovalent association between DCP and dissolved humic substances. Relaxation and diffusion 1H NMR techniques provide rapid and accurate measurements of binding constants and thermodynamic parameters for host−guest complexes between environmental contaminants and natural organic matter

Basis of a Humeomics Science: Chemical Fractionation and Molecular Characterization of Humic Biosuprastructures

We propose a mild stepwise fractionation of molecular components of a humic acid (HA) suprastructure and their structural identification by advanced analytical methods. This procedure may be the basis of a “Humeomics” approach to characterize natural humic molecules and clarify their relations with ecosystems functions. Sequential fractionation included: (1) organic solvent extraction, (2) transesterification with boron trifluoride in methanol (BF3−CH3OH), (3) methanolic alkaline hydrolysis (KOH−CH3OH), and (4) cleavage of ether and glycosidic bonds with HI. Structural identification of initial and final material, separated organo-soluble and hydrosoluble fractions, and subfractions was conducted by GC-MS, HPSEC−ESI−MS (high-resolution, Orbitrap), and solid- and liquid-state NMR. GC-MS revealed in organosoluble unbound fractions the presence of both saturated and unsaturated, linear and branched, alkanoic, hydroxyalkanoic and alkandioic acids, n-alkanes, and n-alkanols. These components decreased progressively in fractions obtained after weak and strong ester cleavage. Unsubstituted alkanoic acids with variable chain length were ubiquitously detected in all fractions, thereby suggesting their fundamental function in the architecture of humic suprastructures. An important role in differentiating supramolecular associations should also be attributed to substituted alkanoic acids that were detected in variable amounts in different fractions. The content of aromatic acids and steroids was only noticed in the latter fractions. HPSEC−ESI−MS of initial and final solid fractions showed similar compounds, as indicated by GC-MS, whereas the hydrosoluble fraction after transesterification revealed fewer of these compounds but noticeable nitrogen-containing acids. A large amount of “cyclic” acids were identified by MS empirical formula in initial HA, and, to a lesser extent, in the final fractionation residue as well as in the hydrosoluble fraction. The predominant alkyl NMR signals in organosoluble extracts and those of CH−N, CH−O, and O−CH−O groups in hydrosoluble fraction confirmed mass spectrometry results. Homo- and heterocorrelated liquid-state NMR spectra indicated spin systems interactions varying with separated fractions. Solid-state and dipolar-dephasing NMR spectra of final residue showed predominance of sp2 carbons, 66% of which were quaternary carbons, and a significant increase in conformational rigidity with respect to initial HA. Separated fractions accounted for 60% of initial HA weight, and losses were attributed to hydration water, liberated volatile compounds, and decarboxylation. Quantization of analytes showed that the sum of compound classes in separated fractions was greater than that for the initial HA, thereby showing that stepwise fractionation increased significantly the analytical identification of humic molecules. Our results suggest this “Humeomics” approach as a valid path for mapping humic molecular composition and assess humus origin and formation

Aggregation and Disaggregation of Humic Supramolecular Assemblies by NMR Diffusion Ordered Spectroscopy (DOSY-NMR)

Diffusion ordered nuclear magnetic resonance spectroscopy (DOSY-NMR) was applied to a number of fulvic (FA) and humic (HA) acids of different origin. Spectral separation achieved by DOSY based on diffusion coefficients (D), and correlated to molecular sizes by calibration standards, showed that carbohydrates had the largest molecular size in FA, whereas alkyl or aromatic components were the most slowly diffusing moieties in HA. At increasing concentrations, these components had invariably lower D values in DOSY spectra for all humic samples, thereby indicating an aggregation into apparently larger associations, whose increased hydrodynamic radius was confirmed by viscosity measurements. When humic solutions were brought from alkaline to acidic pH (3.6), components diffusivity detected by DOSY increased significantly, suggesting a decrease of aggregation and molecular size. A general comparison of HA and FA molecular sizes was achieved by multivariate statistical analysis. While a larger extent of aggregation and disaggregation was observed for HA than for FA, no aggregation was detected, under similar conditions, for a true macropolymeric standard. Such difference in diffusion between a polymeric molecule and humic samples, is in line with the supramolecular nature of humic matter. The possible formation of humic micelles was also investigated by both changes of diffusivity in DOSY spectra and shift of 1H NMR signals. Except for HA of peat and soil origin, revealing a self-assembling in micelle-like structures at the 4 mg mL−1 concentration, no other humic sample showed evidence of critical micelle concentration (cmc) up to 20 mg mL−1. These results indicated that DOSY-NMR spectroscopy is a useful technique to evaluate components of different molecular size in natural humic superstructures

Molecular Characterization of Compost at Increasing Stages of Maturity. 1. Chemical Fractionation and Infrared Spectroscopy

Composted organic biomasses at 60, 90, and 150 days of maturity were studied for changes in molecular composition. Compost samples were subjected to a mild sequential fractionation based on (1) organic solvent extraction, (2) transesterification with boron trifluoride in methanol (BF3−CH3OH), and (3) methanolic alkaline hydrolysis (KOH−CH3OH). The general chemical variations in compost residues following fractionation were monitored by DRIFT spectroscopy, whereas the molecular components separated along the fractionation steps were identified by GC-MS. DRIFT spectra suggested a progressive decrease of biolabile compounds such as alkyls, carbohydrates, and proteinaceous materials with compost maturity. Extraction of unbound components in an organic solvent indicated a considerable reduction of linear and branched alkanoic acids, both saturated and unsaturated, n-alkanes, and n-alkanols with enhancing compost maturity. Extracts of weakly bound molecules by transesterification revealed a decrease, with compost maturity, of components from more recalcitrant plant polyesters, such as ω-, di-, and trihydroxy acids, dioic acids, and n-alkanols. Extracts of strongly bound molecules by alkaline hydrolysis indicated a lower decrease of the same components, suggesting their reduced availability when in stable hydrophobic domains of progressively mature compost. The largest decrease in molecular components occurred when compost was stabilized from 60 to 90 days, whereas its composition did not significantly vary after stabilization at 150 days. The molecular structures of a number of steroids and terpenes appeared to be less susceptible to transformation with composting maturity, thereby resulting as useful biomarkers to trace the fate of composted organic matter in the environment. This work showed that a detailed molecular characterization of compost by a stepwise chemical fractionation enables the evaluation of compost maturity and origin of composted biomasses, as well as the identification of environmental tracers. Keywords: Compost maturity; biomass recycling; molecular characterization; sequential molecular fractionation; hydrophobic component

Quantitative Evaluation of Noncovalent Interactions between Glyphosate and Dissolved Humic Substances by NMR Spectroscopy

Interactions of glyphosate (<i>N</i>-phosphonomethylglycine) herbicide (GLY) with soluble fulvic acids (FAs) and humic acids (HAs) at pH 5.2 and 7 were studied by ¹H and ³¹P NMR spectroscopy. Increasing concentrations of soluble humic matter determined broadening and chemical shift drifts of proton and phosphorus GLY signals, thereby indicating the occurrence of weak interactions between GLY and humic superstructures. Binding was larger for FAs and pH 5.2 than for HAs and pH 7, thus suggesting formation of hydrogen bonds between GLY carboxyl and phosphonate groups and protonated oxygen functions in humic matter. Changes in relaxation and correlation times of ¹H and ³¹P signals and saturation transfer difference NMR experiments confirmed the noncovalent nature of GLY–humic interactions. Diffusion-ordered NMR spectra allowed calculation of the glyphosate fraction bound to humic superstructures and association constants (<i>K</i>_a) and Gibbs free energies of transfer for GLY–humic complex formation at both pH values. These values showed that noncovalent interactions occurred most effectively with FAs and at pH 5.2. Our findings indicated that glyphosate may spontaneously and significantly bind to soluble humic matter by noncovalent interactions at slightly acidic pH and, thus, potentially pollute natural water bodies by moving through soil profiles in complexes with dissolved humus

Oxidative and Photoxidative Polymerization of Humic Suprastructures by Heterogeneous Biomimetic Catalysis

The meso-tetra­(2,6-dichloro-3-sulfonatophenyl)­porphyrinate of manganese­(III) chloride [Mn-(TDCPPS)­Cl] biomimetic catalyst immobilized on spacer-functionalized kaolinite clay mineral was employed in the oxidative coupling reaction of a dissolved humic acid (HA) suprastructure with either chemical (H₂O₂) or UV-light oxidation. The changes in molecular size of humic matter subjected to catalyzed oxidative reaction were followed by high-performance size exclusion chromatography (HPSEC) with UV–vis and refractive index (RI) detectors in series, and by thermogravimetric (TGA) analysis. Both the enhanced molecular size shown by differences between HPSEC chromatograms of humic reaction mixtures at either pH 6 or 3.5 and the increase of thermogravimetric stability suggest that the heterogeneous biomimetic catalysis promoted the stabilization of humic conformations by new intermolecular covalent bonds during oxidative coupling. The similarity between chemical and light-induced oxidation results suggests potential multiple applications of the kaolinite-supported heterogeneous catalyst in controlling the reactivity of natural organic matter within biogeochemical cycles and environmental reactions

High-Resolution Magic-Angle-Spinning NMR and Magnetic Resonance Imaging Spectroscopies Distinguish Metabolome and Structural Properties of Maize Seeds from Plants Treated with Different Fertilizers and Arbuscular mycorrhizal fungi

Both high-resolution magic-angle-spinning (HRMAS) and magnetic resonance imaging (MRI) NMR spectroscopies were applied here to identify the changes of metabolome, morphology, and structural properties induced in seeds (caryopses) of maize plants grown at field level under either mineral or compost fertilization in combination with the inoculation by arbuscular mycorrhizal fungi (AMF). The metabolome of intact caryopses was examined by HRMAS-NMR, while the morphological aspects, endosperm properties and seed water distribution were investigated by MRI. Principal component analysis (PCA) was applied to evaluate ¹H CPMG (Carr-Purcel-Meiboom-Gill) HRMAS spectra as well as several MRI-derived parameters (<i>T</i>₁, <i>T</i>₂, and self-diffusion coefficients) of intact maize caryopses. PCA score-plots from spectral results indicated that both seeds metabolome and structural properties depended on the specific field treatment undergone by maize plants. Our findings show that a combination of multivariate statistical analyses with advanced and nondestructive NMR techniques, such as HRMAS and MRI, enables the evaluation of the effects induced on maize caryopses by different fertilization and management practices at field level. The spectroscopic approach adopted here may become useful for the objective appraisal of the quality of seeds produced under a sustainable agriculture

Oligomerization of Humic Phenolic Monomers by Oxidative Coupling under Biomimetic Catalysis

Three humic phenolic monomers, catechol (CAT), caffeic acid (CAFF), and p-coumaric acid (COUM), were subjected to oxidative coupling catalyzed by biomimetic water−soluble iron−porphyrin (Fe(TDCPPS)Cl) in either separate or mixed solution, and the reaction products were characterized by gas chromatography−mass spectrometry (GC−MS) and electrospray−mass spectrometry (ESI−MS). The GC−MS analysis proved the formation of C−C and C−O dimers, whereas the ESI−MS/MS analysis also suggested trimerization for all the monomers and tetramerization for CAT. On the basis of mass spectra, molecular structures were assigned to the observed oligomers. In the phenolic separate solutions, dimers represented about 65%, 44%, and 30% of reaction products for CAT, CAFF, and COUM, respectively, whereas trimers were from 4 to 5%. A relevant part of the products were unidentified oligomers and several degradation compounds, mostly aromatic aldehydes and alcohols and aromatic or aliphatic carboxylic acids. When all three humic phenolic monomers underwent the catalyzed coupling reaction in one mixed solution, 14% of the reaction products were identified as C−C dimers of CAT. Although no other C−O dimers of CAT, nor any dimers of COUM and CAFF, could be identified, some other structurally unknown oligomers were present among the reaction products of the mixed solution. However, no oligomers larger than tetramers were formed in either separate or mixed solutions. This work indicates the essential role of biomimetic metal−porphyrins in catalyzing the oxidative coupling of humic phenolic monomers in aqueous media, thereby promoting the polymerization of natural organic matter