Synthetic Model Compounds Research Articles

Synthesis of oxidized methyl 4- O-methyl-β- d-glucopyranoside and methyl β- d-glucopyranosyl-(1→4)-β- d-glucopyranoside derivatives as substrates for fluorescence labeling reactions

The synthetic cellulose model compounds methyl 4- O-methyl-β- d-glucopyranoside and methyl 4- O-methyl-β- d-glucopyranosyl-(1→4)-β- d-glucopyranoside and related 6- O-protected intermediates were oxidized in good to fair yields using Swern-conditions or bromine/bis(tributyltin) oxide, respectively, to afford compounds containing 6-aldehyde, 3-keto, and 2,3-diketo groups. Cellobiose and oxidized monosaccharides were then labeled with the carbonyl-selective fluorescence marker 9-(7-amino-1,4,7-trioxaheptyl)-9 H-carbazolecarboxamide (CCOA). The labeled derivatives serve as model compounds for the determination of minute amounts of carbonyl groups in cellulosic polysaccharides.

Synthesis and Testing of a Novel Fluorescence Label for Carboxyls in Carbohydrates and Cellulosics

A carboxyl-selective fluorescence label, 9H-fluoren-2-yl-diaxomethane (FDAM, 6) for the detection of carhoxyl groups specifically in polysaccharides and low-molecular weight carbohydrates has been developed. Attached in apre-column derivatization step, the label allows detection of minute amounts of oxidized functionalities in oligosaccharides and cellulosics under the relatively harsh conditions of GPC separation in the DMAc/LiCl eluant and without any fluorescence interference with light scattering (MALLS) detection, so that carboxyl profiles of polysaccharides were obtained for the first time. Synthetic carbohydrate model compounds (1-3) were used to optimize the labeling reaction.

Single-crystal (57)Fe Q-band ENDOR study of the 4 iron-4 sulfur cluster in its reduced [4Fe-4S](1+) state.

(57)Fe Q-band ENDOR has been used to study the [4Fe-4S](1+) state created by gamma irradiation of single crystals of the synthetic model compound [N(C(2)H(5))(4)](2)[Fe(4)S(4)(SCH(2)C(6)H(5))(4)] enriched in (57)Fe. This compound is an excellent biomimetic model of the active sites of many 4 iron-4 sulfur proteins, enabling detailed and systematic studies of its oxidized [4Fe-4S](3+) and reduced [4Fe-4S](1+) paramagnetic states. Taking advantage of the fact that Q-band ENDOR, in contrast with X-Band ENDOR, allows for a very good separation of the (57)Fe transitions from those of the protons, the complete hyperfine tensors of the four iron atoms for the [4Fe-4S](1+) species has been measured with precision. For each iron atom, the electron orbital and electron spin isotropic contributions have been determined separately. Moreover, it is remarkable that two (57)Fe hyperfine tensors attributed to the ferrous pair of iron atoms are very different. In effect, one tensor presents a much larger anisotropic part and a much smaller isotropic part than those of the other. This difference has been interpreted in terms of a differential electron orbital hyperfine interaction among the two ferrous ions.

Detailed Proton Q-Band ENDOR Study of the Electron Spin Population Distribution in the Reduced [4Fe-4S]1+ State

Proton ENDOR spectroscopy has been used in single crystals of the synthetic model compound [N(C2D5)4]2[Fe4S4(SCH2C6D5)4] in order to determine the electron spin population distribution within the [4Fe-4S]1+ paramagnetic state. This compound constitutes an excellent biomimetic model of the active sites of many [4Fe-4S] proteins, with the [4Fe-4S]1+ state corresponding to the reduced state of ferredoxin proteins and of numerous other related proteins. Because the information obtained by ENDOR and (or) by NMR from these protons constitutes the basis of the most detailed knowledge on these proteins from magnetic resonance methods, our purpose is to set a firm basis for their interpretation. Only by using Q-band ENDOR could we succeed in measuring the complete hyperfine tensors of a total of 11 protons, i.e., the eight protons of the four CH2 groups and three additional protons from nearest neighbors. By appealing to a new version of the multicentric point-dipole analysis of the dipolar parts of these tensors, we could deduce a detailed map of the electron spin population distribution. This new approach is based on the direct determination of the four spin projection coefficients centered on the four iron monomers constituting the cluster. Our findings confirm that two classes of reduced [4Fe-4S]1+ have to be distinguished, according to the magnitude of the spin coupling coefficients that best describe their fundamental magnetic spin states. Such a distinction has been observed already among oxidized [4Fe-4S]3+ centers. This opens the way for further research aiming at understanding the relations between structures, redox states, ligation, and magnetic spin states.

ChemInform Abstract: Amphidinolides T2, T3, and T4, New 19‐Membered Macrolides from the Dinoflagellate Amphidinium sp. and the Biosynthesis of Amphidinolide T1.

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Amphidinolides T2, T3, and T4, new 19-membered macrolides from the dinoflagellate Amphidinium sp. and the biosynthesis of amphidinolide T1.

Three new 19-membered macrolides, amphidinolides T2 (2), T3 (3), and T4 (4), structurally related to amphidinolide T1 (1) have been isolated from two strains of marine dinoflagellates of the genus Amphidinium. The structures of 2-4 were elucidated on the basis of spectroscopic data. The absolute configurations at C-7, C-8, and C-10 of 1-4 were determined by comparison of NMR data of their C-1-C-12 segments with those of synthetic model compounds for the tetrahydrofuran portion. The biosynthetic origins of amphidinolide T1 (1) were investigated on the basis of 13C NMR data of a 13C enriched sample obtained by feeding experiments with [1-(13)C], [2-(13)C], and [1,2-(13)C2] sodium acetates and 13C-labeled sodium bicarbonate in the cultures of the dinoflagellate. These incorporation patterns suggested that amphidinolide T1 (1) was generated from four successive polyketide chains, an isolated C1 unit formed from C-2 of acetates, and three unusual C2 units derived only from C-2 of acetates. Furthermore, it is noted that five oxygenated carbons of C-1, C-7, C-12, C-13, and C-18 were not derived from the C-1 carbonyl, but from the C-2 methyl of acetates.

Carbon-13 Solid-State NMR Studies on Synthetic Model Compounds of [4Fe−4S] Clusters in the 2+ State

Carbon-13 solid-state NMR is used to determine, via the temperature dependence of paramagnetic shifts, the spin ladder of magnetic levels and the related spin exchange parameters in [4Fe-4S](2+) clusters. This study has been carried out between 180 and 330 K in three model compounds of the active sites of 4Fe-4S proteins: [(C2D5)(4)N](2)[Fe4S4((SCD2C6D5)-C-13)(4)], [(C2H5)(4)N](2)[Fe4S4(SC(CH3)(3))(4)], and [(C2H5)(4)N](2)[Fe4Se4(SC-(CH3)(3))(4)] The temperature dependencies of both the isotropic and anisotropic parts of the C-13 paramagnetic shift tensors have been measured and analyzed. Antiferromagnetic exchange coupling constants among iron atoms of J approximate to 430, 380, and 370 cm(-1) have been deduced from fits of their temperature dependencies. Additional contributions of the double-exchange term B favoring electronic delocalization and of a DeltaJ term associated with the compression of the cubane geometry are also discussed. We also show that connections between these measurements and those of magnetic susceptibility are especially close in the case of the [Fe-4-4S](2+) odor state.

Transformation and mineralization of synthetic 14C-labeled humic model compounds by soil-feeding termites

The majority of termite species are considered to be humivorous, but the exact nature of their carbon and energy source, the mechanisms involved in digestion and the impact of this feeding habit on the humification of soil organic matter are largely obscure. We performed feeding trials with soil-feeding termites (Termitidae: Termitinae), using 14C-labeled humic model compounds as substrates. In the case of Cubitermes orthognathus, the components of chemically identical synthetic humic acids (HA), labeled either in their proteinaceous or aromatic building blocks, and synthetic fulvic acids (FA), labeled in the aromatic building blocks, all had similarly low mineralization rates (2.5, 2.2 and 2.7%, respectively) when incubated in soil for 10 days in the absence of termites. When termites were present, the mineralization rate of the aromatic component of HA and FA increased only slightly but significantly (2.4 and 3.1%, respectively; P < 0.05), whereas that of the proteinaceous component increased more than 10-fold (30.8%). Similar results were obtained when the protein was peptonized prior to polymerization (9.7 vs. 27.4% mineralization). Mineralization of HA was accompanied by a transformation of the residual peptide label to FA, whereas the aromatic label of HA and FA was partly transformed to humin during gut passage. High-performance gel permeation chromatography showed a strong shift in the size-class distribution of peptide label towards low-molecular-weight products, especially of the material ingested by the termites; the smallest molecules were recovered from the termite bodies. Similar results were obtained in feeding trials with Cubitermes umbratus and Thoracotermes macrothorax. Together with previous findings, the current results provide strong evidence that during gut passage, the combined action of extreme alkalinity in the anterior hindgut, autoxidative processes, and probably also proteolytic activities, renders a large reservoir of potential substrates accessible. While peptidic components of humic substances are selectively digested, aromatic components are apparently not an important food source for soil-feeding termites. These findings have important implications for the mobilization of organic nitrogen in tropical soils.

Synthesis and characterization of specifically 14C-labeled humic model compounds for feeding trials with soil-feeding termites

The feeding activity of soil-feeding termites is considered an important factor in the turnover of soil organic matter in tropical ecosystems, but the nature of the components exploited as carbon and energy source and the consequences of the gut passage for the residual fraction are still largely obscure. In order to determine mineralization rates of specific components in feeding experiments, we prepared 14C-labeled synthetic humic model compounds by peroxidase-initiated radical polymerization of a mixture of phenolic compounds, peptides, amino acids, and carbohydrates. A general characterization of the polymerization products showed that they resembled natural humic substances prepared from tropical soil used in feeding trials with soil-feeding termites in their elemental content, infrared spectra, and molecular weight distribution. Solubility-based fractionation of chemically identical preparations, arising from selective labeling of different precursors, revealed that almost all of the label stemming from the protein precursors and approximately two-thirds stemming from the peptone and catechol precursors were recovered in the humic acid fraction. In contrast, the label of glucose or glycine was recovered almost completely in the acid-soluble fraction. High-performance gel-permeation chromatography (HP-GPC) of the humic and fulvic acid fractions combined with online radiotracer analysis showed large differences in the size distribution of the radiolabel, which depended strongly on the chemical nature of labeled precursor.

Exchange variation of zero-field splittings in [Fe 4S 4] + clusters of ferredoxins with high-spin S=3/2 ground state

The spin-coupling model of zero-field splitting (ZFS) of tetrameric mixed-valent and monovalent clusters is developed. The 〈| s ̂ i0 (2)|〉 matrix elements of the individual ZFS tensor operator of the second rank were calculated for tetramers of the general type in the representation of the total and intermediate spins. The spin-dependent correlations between ZFS parameters D S of the cluster states S and D i of individual ions s i were obtained for [4Fe–4S] + centers of native systems and synthetic model compounds with the high-spin S gr=3/2 ground state. It was shown that the single-particle spin parameters f i=〈| s ̂ iz 2−s i(s i+1)/3|〉/〈| S ̂ z 2−S(S+1)/3|〉 of the [Fe 3(II)Fe(III)] center essentially depend on the total, intermediate and local spins and on the exchange parameters. In the spin schemes with fixed intermediate spins, the correlation between the cluster total spin ZFS parameters, D S , and the individual ZFS parameters, D i , are determined by the total, intermediate and individual spins. Anisotropic cluster ZFS parameters D S strongly depend on isotropic Heisenberg exchange and double exchange inter-ion interactions due to exchange mixing of the states with different intermediate spins. It was shown that the cluster ZFS parameters D S , for the states with S=3/2, change their values and sign under the variation of the Heisenberg exchange and double exchange parameters during the cluster deformation. The single-particle ZFS parameters D i and the exchange effects determine the observed strong positive ( D S =1.5–6 cm −1) and negative ( D S =−1.7 to −5 cm −1) cluster ZFS splittings of the ground S gr=3/2 states of the [4Fe–4S] + clusters. Positive (negative) cluster ZFS parameters D S correspond to negative (positive) individual ZFS parameters D 1−3 [Fe(II)]. The theory explains the observation of small negative effective hyperfine constants A i for the S gr=3/2 clusters with positive and negative cluster ZFS parameters D S . The correlations between the individual and cluster ZFS parameters were obtained for mixed-valent high-spin [4Fe–4S] + clusters with S gr=5/2, 7/2 and monovalent [4Fe–4S] 0 cluster with S gr=4.

Inactivation of cysteine proteases by peptidyl epoxides: characterization of the alkylation sites on the enzyme and the inactivator

Erythro peptidyl epoxides are selective inactivators of cysteine proteases. The alkylation site, both on the enzyme papain and on the epoxide itself, was characterized. The inactivation of papain with the peptidyl epoxide erythro benzyloxycarbonyl-Phe-Ala-epoxide was followed by total hydrolysis by acid. Mass spectral analysis of the hydrolysate revealed, in addition to the expected amino acids, a unique signal of m/z 209 (MH(+)). Its high-resolution mass spectrum and daughter peak analysis correspond to the product of alkylation on cysteine and the expected fragmentation. A similar MS pattern was obtained for a synthetic model compound corresponding to the expected hydrolysis product. A (13)C NMR analysis of papain inactivated by a specifically (13)C-labelled peptidyl epoxide indicated that the alkylation of the enzyme's cysteine residue occurs on the primary carbon of the epoxide moiety.

Carbon-13 and Oxygen-17 Chemical Shifts, (16O/18O) Isotope Effects on13C Chemical Shifts, and Vibrational Frequencies of Carbon Monoxide in Various Solvents and of the Fe−C−O Unit in Carbonmonoxy Heme Proteins and Synthetic Model Compounds

13C shieldings, δ(13C), 17O shieldings, δ(17O), and 18O isotope effects on 13C shieldings, 1Δ13C(18O/16O), of carbon monoxide (99.7% 13C, 0.9% 17O, and 11.8% 18O enriched) in a variety of solvents and of the Fe−C−O unit of several carbonmonoxy hemoprotein models with varying polar and steric effects of the distal organic superstructure and constraints of the proximal side are reported. This enables, first, comparisons with hemoproteins, C−O vibrational frequencies, ν(C−O), and X-ray structural data to be made; second, to investigate whether polarizable CO is an adequate model for distal ligand effects in carbonmonoxy heme proteins and synthetic model compounds; third, to investigate the effect of electronic perturbation within the heme pocket and pocket deformation on δ(13C), δ(17O), 1Δ13C(18O/16O), and ν(C−O). A variety of solvents with varying dielectric constants and solvation abilities appears to have negligible effect on δ(17O), δ(13C), and 1Δ13C(18O/16O) and little direct effect on ν(C−O) of dissolv...

Amphilactams A−D: Novel Nematocides from Southern Australian Marine Sponges of the Genus Amphimedon

Bioassay-directed fractionation of the ethanol extracts of two Amphimedon spp. collected during trawling operations in the Great Australian Eight yielded four new macrocyclic lactone/lactams, amphilactams A-D (1-4). The amphilactams possess potent in vitro nematocidal properties, and their structures were assigned on the basis of detailed spectroscopic analysis and comparison with synthetic model compounds. The amphilactams feature both carbon skeletons and an enamino lactone/lactam moiety unprecedented in the natural products literature.

X-ray Spectroscopy of Nitric Oxide Binding to Iron in Inactive Nitrile Hydratase and a Synthetic Model Compound

Analysis of the iron K-edge X-ray absorption spectrum (XAS) of the “dark”, inactive form of nitrile hydratase (NHdk) from Rhodococcus sp. R312 confirms a 1:1 stoichiometry of nitric oxide bound to low-spin iron(III). We also report XAS analyses of four iron complexes of the pentadentate ligands 2,3,13,14-tetramethyl-4,8,12-triaza-3,12-pentadecadiene-2,14-dithiolate (L2-, also denoted as S2Me2N3(Pr,Pr)2-) and 2,12-dimethyl-3,7,11-triaza-2,11-tridecadiene-1,13-dithiolate (L‘2-, also denoted as S2N3(Pr,Pr)2-): five-coordinate FeIIL‘ and FeL+ and low-spin six-coordinate FeL(N3) and FeL(NO)+ (cationic species are PF6- salts). The XAS of FeL(N3) and FeL(NO)+ closely mimic the spectra of butyrate-stabilized active nitrile hydratase (NHlt) and NHdk, respectively. The 1s → 3d pre-edge peak is about twice as intense in five-coordinate FeL+ than for the remaining samples, suggesting that the iron in both NHlt and NHdk is six-coordinate. This peak and other edge features are 1 eV higher in energy for NHdk and FeL(NO...

Feruloyl Esters of ω-Hydroxyacids in Cork Suberin

Feruloyl esters of ω-hydroxyacids were found in the alcoholysis products of suberin from Quercus suber cork. Their identification was made from mass spectra and by comparison with synthetic model compounds. The co-hydroxyls of suberinic hydroxyacids are thought to be the ester-bonding points between suberin aliphatics and associated aromatics.

Identification of isomeric tributylphosphate dimers formed by radiolysis using tandem mass spectrometry and stable isotopic labelling

Tributylphosphate (TBP) dimers, formed during radiolysis of the nuclear fuel reprocessing solvent, were studied using tandem mass spectrometry (MS/MS), stable isotopic labelling ( 2 H) and synthetic model compounds. The structures of isomeric dimers and their lower homologues were established, using GC/MS as well as electron impact and chemical ionisation GC/tandem mass spectrometry, with an extensive use of ion chromatograms of characteristic fragment ions from collision-induced decomposition in both nondeuterated and selectively deuterated series. An explanation of isomer ratios and of the restricted number of lower homologues as well as a radiolytic mechanism are advanced. The TBP-dimer fraction represented less than ~ 0.03% of the total mixture obtained from radiolysis of solvent.

Characterization of the Native Lysine Tyrosylquinone Cofactor in Lysyl Oxidase by Raman Spectroscopy

Lysine tyrosylquinone (LTQ) recently has been identified as the active site cofactor in lysyl oxidase by isolation and characterization of a derivatized active site peptide. Reported in this study is the first characterization of the underivatized cofactor in native lysyl oxidase by resonance Raman (RR) spectrometry. The spectrum is characterized by a unique set of vibrational modes in the 1200 to 1700 cm-1 region. We show that the RR spectrum of lysyl oxidase closely matches that of a synthetic LTQ model compound, 4-n-butylamino-5-ethyl-1,2-benzoquinone, in aqueous solutions but differs significantly from those of other topa quinone-containing amine oxidases under similar conditions. Furthermore, we have observed the same 18O shift of the C=O stretch in both the lysyl oxidase enzyme and the LTQ cofactor model compound. The RR spectra of different model compounds and their D shifts give additional evidence for the protonation state of LTQ cofactor in the enzyme. The overall similarity of these spectra and their shifts shows that the lysyl oxidase cofactor and the model LTQ compound have the same structure and properties. These data provide strong and independent support for the new cofactor structure, unambiguously ruling out the possibility that the structure originally reported had been derived from a spurious side reaction during the derivatization of the protein and isolation of the active site peptide.

Paramagnetic States of Four Iron−Four Sulfur Clusters. 2. Proton ENDOR Study of a 1+ State in an Asymmetrical Cluster

Proton ENDOR spectroscopy on γ-irradiated single crystals of the synthetic model compound (Et4N)2[Fe4S4(SC6H4-o-OH)4] has been used to study a [4Fe−4S]+ center that has properties similar to those of the reduced states of some particular ferredoxins and of the enzyme aconitase. The interesting point about this model compound is that one iron atom is five-coordinate, with an extra phenolic oxygen attached to one of its thiolate ligand. The complex thus represents an example of an asymmetrical [4Fe−4S] cluster in the solid crystalline state. From the study of the angular dependence of the proton ENDOR lines, it has been possible to derive eight hyperfine tensors of protons occupying different positions in the near vicinity of the iron atoms, the main bearers of the spin population of the paramagnetic anion. Some protons belong to thiolate ligands of the anion whereas others belong to close (Et4N)+ counterions. From the anisotropic parts of the tensorsand by using a multicentric point-dipole modelit has been...

DNA cleavage by iron(III) complex of synthetic bleomycin model compound

DNA cleavage by iron(III) complex of synthetic bleomycin model compound

Molecular Characterization of Cr Phases in Contaminated Soils by Cr and Fe EXAFS : A Tool for Evaluating Chemical Remediation Strategies

The successful remediation of Cr contaminated soils via chemical reduction and stabilization of soluble, toxic Cr(VI) species to insoluble Cr(III) species relies on the long term leachability and reoxidation of reduced Cr compounds. Information of Cr chemical environments is invaluable for evaluating the efficiency of proposed stabilization technologies. We describe a matrix of Fe and Cr K-edge EXAFS data on synthetic model compounds that have been used to identify different Fe and Cr local environments of two soil samples.