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Made available in DSpace on 2021-06-25T10:50:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-04-01 John Innes Foundation Seventh Framework Programme European Regional Development Fund Fonds Wetenschappelijk Onderzoek Plant roots are specialized belowground organs that spatiotemporally shape their development in function of varying soil conditions. This root plasticity relies on intricate molecular networks driven by phytohormones, such as auxin and jasmonate (JA). Loss-of-function of the NOVEL INTERACTOR OF JAZ (NINJA), a core component of the JA signaling pathway, leads to enhanced triterpene biosynthesis, in particular of the thalianol gene cluster, in Arabidopsis thaliana roots. We have investigated the biological role of thalianol and its derivatives by focusing on Thalianol Synthase (THAS) and Thalianol Acyltransferase 2 (THAA2), two thalianol cluster genes that are upregulated in the roots of ninja mutant plants. THAS and THAA2 activity was investigated in yeast, and metabolite and phenotype profiling of thas and thaa2 loss-of-function plants was carried out. THAA2 was shown to be responsible for the acetylation of thalianol and its derivatives, both in yeast and in planta. In addition, THAS and THAA2 activity was shown to modulate root development. Our results indicate that the thalianol pathway is not only controlled by phytohormonal cues, but also may modulate phytohormonal action itself, thereby affecting root development and interaction with the environment. Department of Plant Biotechnology and Bioinformatics Ghent University, Technologiepark 71 VIB Center for Plant Systems Biology, Technologiepark 71 Department of Metabolic Biology John Innes Centre, Norwich Research Park, Colney Lane Laboratory of Molecular Cell Biology KU Leuven, Kasteelpark Arenberg 31 VIB Center for Microbiology, Kasteelpark Arenberg 31 Department of Organic Chemistry Institute of Chemistry São Paulo State University (UNESP) Laboratory of Growth Regulators Institute of Experimental Botany of the Czech Academy of Sciences and Faculty of Science of Palacký University, Šlechtitelů 27 Department of Organic Chemistry Ghent University Department of Forest Genetics and Plant Physiology Umeå Plant Science Centre Swedish University of Agricultural Sciences Lab of Plant Growth Analysis Ghent University Global Campus Department of Organic Chemistry Institute of Chemistry São Paulo State University (UNESP) Seventh Framework Programme: 613692 TriForC European Regional Development Fund: CZ.02.1.01/0.0/0.0/17_048/0007323 Fonds Wetenschappelijk Onderzoek: G004515N Fonds Wetenschappelijk Onderzoek: G008417N

Pseudomonas isolates from tropical environments have been underexplored and may form an untapped reservoir of interesting secondary metabolites. In this study, we compared Pseudomonas and cyclic lipopeptide (CLP) diversity in the rhizosphere of a cocoyam root rot disease (CRRD) suppressive soil in Boteva, Cameroon with those from four conducive soils in Cameroon and Nigeria. Compared with other soils, Boteva andosols were characterized by high silt, organic matter, nitrogen and calcium. Besides, the cocoyam rhizosphere at Boteva was characterized by strains belonging mainly to the P. koreensis and P. putida (sub)groups, with representations in the P. fluorescens, P. chlororaphis, P. jessenii and P. asplenii (sub)groups. In contrast, P. putida isolates were prominent in conducive soils. Regarding CLP diversity, Boteva was characterized by strains producing 11 different CLP types with cocoyamide A producers, belonging to the P. koreensis group, being the most abundant. However, putisolvin III-V producers were the most dominant in the rhizosphere of conducive soils in both Cameroon and Nigeria. Furthermore, we elucidated the chemical structure of putisolvin derivatives-putisolvin III-V, and described its biosynthetic gene cluster. We show that high Pseudomonas and metabolic diversity may be driven by microbial competition, which likely contributes to soil suppressiveness to CRRD.

A novel technique based on high resolution (HR)-NMR diffusometry is presented to investigate oil diffusion on the (sub)-micron scale level in fat crystal networks in situ and in a non-destructive way. The use of a high-gradient probe enables the observation of the time (Delta)-dependent diffusion behavior in fat blends (FBs) of different solid fat content (SFC), as obtained by mixing soft palm mid fraction and high-oleic sunflower oil. Hereby, the NMR-diffusion data are analyzed using CONTIN to obtain a continuous distribution of diffusion coefficients. As such, the hindrance of the oil diffusion by the fat crystal network is examined. The characterization of the lipid matrices is completed with H-1-LR-NMR relaxometry, X-ray diffraction, and microstructural visualization. For the investigated FBs, the average diffusion coefficient D(Delta) decreased with increasing SFC, indicating an increased tortuosity of the diffusive path due to an increasing density of the fat crystal networks as observed by polarized light microscopy. This is also supported by an increase in crystal surface area which is indicated by T-2-relaxation analysis. This study shows that NMR diffusometry is a promising technique for investigation of structure-function relationships in fat systems. Practical Applications: The study shows that NMR diffusometry is a promising technique for investigation of structure-function relationships in fat systems. For the first time, oil diffusion in fat crystal networks has been studied by HR-NMR diffusometry with high gradients to collect the diffusional echo signals, while CONTIN-analysis has been used for the first time to process the data.

The structural importance of backbone amide protons in the bacterial cyclic lipopeptide pseudodesmin A, a viscosin group member, is studied by selective introduction of N-methylated amino acids. Site-selective N-methylation was conveniently achieved by introduction of N-Me-L-Leu at positions 1 and 7 during solid phase peptide synthesis. The impact of this modification on conformation, antibiotic activity and supramolecular behaviour allowed to establish the significance of intra- and intermolecular hydrogen bonds.

Bola-amphiphilic surfactants are molecules with fascinating properties. Their unique configuration consisting of a long hydrophobic spacer connecting two hydrophilic entities renders the molecule more water soluble than the average surfactant, but still allows formation of supramolecular structures. These properties make them extremely suitable for applications in in nanotechnology, electronics, and gene and drug delivery. In general, these compounds are obtained by chemical synthesis. We report here an efficient microbial production process for the fully green synthesis of bolaform surfactants. A sophorolipid-producing Starmerella bombicola yeast strain was disabled in its sophorolipid acetyltransferase and lactone esterase, which should logically result in synthesis of non-acetylated acidic sophorolipids; molecules with the classic amphiphilic structure. Surprisingly, also bolaform glycolipids were obtained, with an additional sophorose linked to the free carboxyl end of the acidic sophorolipids as confirmed by MS and NMR analysis. The obtained titers of 27.7 g/L total product are comparable to wild type values, and the novel molecules account for at least 74% of this. Bola-amphiphile biosynthesis proved to be attributed to the promiscuous activity of both UDP-glucosyltransferases UGTA1 and UGTB1 from the core sophorolipid pathway, displaying activity toward non-acetylated intermediates. The absence of acetyl groups seems to trigger formation of bolaform compounds starting from acidic sophorolipids. Hence, wild type S. bombicola produces these compounds only at marginal amounts in general not reaching detection limits. We created a strain knocked-out in its sophorolipid acetyltransferase and lactone esterase able to produce these novel compounds in economical relevant amounts, opening doors for the application of biological-derived bolaform structures. Biotechnol. Bioeng. 2016;113: 2644-2651. © 2016 Wiley Periodicals, Inc.

Cyclic lipodepsipeptides (CLPs) are a group of metabolites produced by Pseudomonas bacteria, involved in various biological functions and displaying a wide range of properties, including antibacterial and antifungal activities. The white line-inducing principle (WLIP) is a member of the viscosin group featuring a Glu2 amino acid. Recently, a total synthesis of pseudodesmin A - the Gln2 counterpart of WLIP - was described, and we here expand this route to Glu2 containing CLPs. We report the first total synthesis of WLIP and at the same time establish that the Gln2 to Glu2 substitution has an adverse impact on the crude purity and overall yield. A comparative study of different CLP analogues reveals the importance of the nature of the Glx2 protecting group in determining these outcomes. Replacement of the conventional tBu protecting group by the larger benzyl group for the Glu residue in our synthesis strategy indeed resulted in an improved conversion. Next to achieving the first WLIP total synthesis, we thus show the importance of a careful choice of protecting groups for the success of this type of solid-phase synthesis approaches towards CLPs.

Seed survival in soil could be strongly influenced by habitat characteristics, but little is known about the behaviour of seeds sensitive to desiccation in seed banks installed in natural or disturbed habitats. Cryptocarya aschersoniana seeds disperse at the end of the rainy season but do not germinate immediately; thus, they may form seed banks in soil. This study evaluated the behaviour of C. aschersoniana seed banks induced in the natural environment of the species and in a disturbed area. Recently harvested C. aschersoniana germination units were characterized according to their water content, germination and viability. In 2011 and 2012, seed banks were established by burying samples of seeds in the understory of a semi-deciduous forest. In 2012, samples were also buried in a disturbed area. The seed banks were sampled at certain time intervals, and the samples were characterized as described above. Precipitation and air temperature data were collected. As a result, seeds in the seed bank established in the natural environment form a transient seed bank and showed the same behaviour in both years studied. A germination peak was observed starting 210 days after burial (coinciding with the onset of the rainy season) and reached germination percentages higher than 80% at the end of the experiment for both years. Seed mortality did not exceeded 28% in the natural environment. However, in the disturbed environment, the seeds lost their viability more rapidly, with 90% of the seeds becoming unviable 240 days after burial. Germinated seeds in the disturbed environment (maximum 21%) were not able to establish seedlings. These results underscore the importance of maintaining a natural, undisturbed forest for the conservation of this species.

Biocompatible inorganic nano- and microcarriers can be suitable candidates for protein delivery. This study demonstrates facile methods of functionalization by using nanoscale linker molecules to change the protein adsorption capacity of hydroxyapatite (HA) powder. The adsorption capacity of bovine serum albumin as a model protein has been studied with respect to the surface modifications. The selected linker molecules (lysine, arginine, and phosphoserine) can influence the adsorption capacity by changing the electrostatic nature of the HA surface. Qualitative and quantitative analyses of linker-molecule interactions with the HA surface have been performed by using NMR spectroscopy, zeta-potential measurements, X-ray photoelectron spectroscopy, and thermogravimetric analyses. Additionally, correlations to theoretical isotherm models have been calculated with respect to Langmuir and Freundlich isotherms. Lysine and arginine increased the protein adsorption, whereas phosphoserine reduced the protein adsorption. The results show that the adsorption capacity can be controlled with different functionalization, depending on the protein-carrier selections under consideration. The scientific knowledge acquired from this study can be applied in various biotechnological applications that involve biomolecule-inorganic material interfaces.

Ligand exchange is central in the processing of inorganic nanocrystals (NCs) and requires understanding of surface chemistry. Studying sterically stabilized HfO2 and ZrO2 NCs using (1) H solution NMR and IR spectroscopy as well as elemental analysis, this paper demonstrates the reversible exchange of initial oleic acid ligands for octylamine and self-adsorption of oleic acid at NC surfaces. Both processes are incompatible with an X-type binding motif of carboxylic acids as reported for sulfide and selenide NCs. We argue that this behavior stems from the dissociative adsorption of carboxylic acids at the oxide surface. Both proton and carboxylate moieties must be regarded as X-type ligands yielding a combined X2 binding motif that allows for self-adsorption and exchange for L-type ligands.

A rapid and efficient total synthesis is reported for the cyclic lipodepsipeptide pseudodesmin A. This member of the Pseudomonas viscosin group is active against Gram-positive bacteria and features self-assembling properties. A conserved serine residue within the lactone macrocycle is exploited for initial immobilization on 2-chlorotrityl chloride resin through ether formation with the side-chain alcohol. Subsequent elongation proceeds through Fmoc solid-phase peptide synthesis, including automated incorporation of the enantioselectively synthesized (R)-3-hydroxydecanoic acid lipid tail. Following esterification to generate the incipient lactone bond, the macrocycle is formed by on-resin head-to-tail macrolactamization and cleaved from the resin to give the desired compound in good purity. The short and efficient synthesis route allows rapid generation of analogues by facile variation of both the peptide and lipid moieties with good control of epimerization while maximizing automation. Synthesis of the pseudodesmin A enantiomer yields identical self-assembly and biological activity to that observed for the natural compound, showing that activity is not mediated by chiral interactions. A D-Asn8 analogue developed en route retains self-assembly, but loses activity. The synthesis strategy should be generally applicable for the rapid generation of analogues from various cyclic lipodepsipeptide groups, allowing an investigation of their self-assembling properties and structure-activity relationships.

The synthesis and evaluation of gelatin acrylamide as an alternative to the well-established gelatin methacrylamide are reported. High-resolution magic angle spinning NMR is used for the quantitative determination of the chemical cross-linking density. The gelatin acrylamide precursors reveal enhanced cross-linking in terms of reactivity and double bond conversion, resulting in stronger networks. Remarkably, even at very low double bond conversions, ≈5%, gel fractions of ≈40% are obtained. The cross-linked networks are also reviewed in the framework of the rubber elasticity and thermodynamic swelling theories to estimate important nanostructural properties. Preliminary cell tests revealed that highly viable (>90%) cell-laden constructs are obtained.

We present the use of 1-mm room-temperature probe technology to perform intermolecular interaction studies using chemical shift perturbation methods and saturation transfer difference (STD) spectroscopy using small sample volumes. The use of a small sample volume (5–10 µl) allows for an alternative titration protocol where individual samples are prepared for each titration point, rather than the usual protocol used for a 5-mm probe setup where the ligand is added consecutively to the solution containing the protein or host of interest. This allows for considerable economy in the consumption and cost of the protein and ligand amounts required for interaction studies. For titration experiments, the use of the 1-mm setup consumes less than 10% of the ligand amount required using a 5-mm setup. This is especially significant when complex ligands that are only available in limited quantities, typically because they are obtained from natural sources or through elaborate synthesis efforts, need to be investigated. While the use of smaller volumes does increase the measuring time, we demonstrate that the use of commercial small volume probes allows the study of interactions that would otherwise be impossible to address by NMR. Copyright © 2010 John Wiley & Sons, Ltd.

The isolation and spectrometric characterization of mutumol, 22-acetoxy-3β,23-dihydroxy-24-methylenelanost-8-ene, from the fungus Pisolithus arhizus (Pers.) Rausch is described. Besides the usual spectrometric methods, also an X-ray crystallographic analysis and a complete 1H 500 MHz NMR analysis was performed.

Chemical modification reactions of alkyne containing polyHEMA-based macroporous network structures (cryogels) by Cu(I) catalyzed azide-alkyne 'click' cycloaddition reactions and their monitoring and quantification with high-resolution magic angle spinning (hr-MAS) NMR spectroscopy are reported. Complete conversion is obtained when benzylazide is reacted with the grafted alkyne function, but only partial conversion is observed when using azide-modified poly(ethylene glycol) (PEG-N(3) ). Subsequent addition of benzylazide consumes all remaining alkyne groups. All chemical modifications are easily monitored at each stage using hr-MAS NMR spectroscopy. The alkyne functionality and the resulting triazole ring provide well resolved (1) H resonances to monitor and quantify the progress of such 'click' reactions in general.

Second-generation catalysts of the general formula Cl2Ru(SIMes)(L)(3-phenylinden-1-ylidene), 3a (L = PCy3), 3b (L = PPh3), 3c (L = py), and Cl2Ru(SIMe)(L)(3-phenylinden-1-ylidene), 4a (L = PCy3), 4b (L = PPh3), 4c (L = py) were found to be of interest in various metathesis transformations. The catalysts containing SIMe ligands showed improved initiation compared to the more robust SIMes substituted catalysts. A strong temperature effect was noted on all of the reactions tested. Interestingly, complex 3a, showing the lowest initiation rate at room temperature, emerged as the most productive of all systems examined at elevated temperature. It is shown that complexes containing the SIMe ligand display higher initiation efficiency than their corresponding SIMes analogues. Since the higher initiation is related to the ease of phosphane dissociation while phosphane dissociation also promotes catalyst decomposition, complexes bearing the SIMe ligand decompose faster. The complete 1H, 13C and 31P resonance assignment and the procedure applied to obtain these from a combination of 1D and 2D NMR techniques, is also reported. Combined with the ROESY technique, these enabled to investigate several conformational processes involving rotations around N–phenyl and C–Ru bonds on the millisecond to second timescale. A clear correlation is demonstrated between the bulkiness of the axial ligand (L) and the rotational freedom of the SIMe(s) ligand. A qualitative analysis also suggests that the extra para-methyl of SIMes leads to additional steric interactions with the 3-phenylinden-1-ylidene ligand. The data reported in this paper demonstrates that substitution patterns of the N-aryl have a significant influence on the activity of the second-generation indenylidene catalysts for a given metathesis reaction.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Solution NMR spectroscopy is a well established technique for nondestructive characterization of the structures and conformations of complex oligo- and polysaccharides. One of the key experiments involves the use of 2D TOCSY to collect the 1 H spins into groups that can be associated with the individual saccharide units that are present in the molecule under study. It is well known that the magnetization transfer rate through the 1 H spin system during the TOCSY spin lock period is sensitive to the intervening 3 J-(H,H) scalar couplings, and therefore also to the saccharide stereochemistry. Here, we have investigated the potential to extract information on the stereochemistry of hexapyranose monosaccharide units directly from TOCSY spectra. Through a systematic experimental investigation of the magnetization transfer initiated from the anomeric 1 H resonance in D-glucose, D-galactose and D-mannose it is shown that a 100 ms spin lock time provides optimal spectroscopic discrimination between these three commonly occurring building blocks. A simple matching scheme is proposed as a new tool for rapid attribution of the TOCSY traces originating from the anomeric 1 H resonances towards the underlying monosaccharide type. The scheme appears robust with regard to structural variations and fairly tolerant to incidental overlap. Its application provides useful guidance during the subsequent NMR assignment process, as demonstrated with the PS7F polysaccharide from Streptococcus pneumonia. In addition, we show that our scheme affords a clear-cut distinction between the α-and β-epimers of D-mannose-type units, which can be difficult to discriminate by NMR analysis. Application to the N-glycan 100.2 demonstrates the potential and wide applicability of this new discrimination approach.

Diffusion-ordered NMR spectroscopy (DOSY NMR) is presented as a tool for the determination of the diffusion coefficients of organic ligands in suspensions of titanium dioxide nanoparticles. The nanoparticles were prepared by a sol-gel process by hydrolysis and condensation reactions of titanium tetra-n-butoxide in the presence of pentane-2,4-dione (acacH: acetylacetone), as well as para-toluenesulfonic acid (pTsA) and n-butanol (nBuOH). NMR spectroscopic studies were performed in various deuterated solvents, on both dispersed xerosols and diluted sols. The bipolar-pulsed field gradient longitudinal eddy-current delay (LED) pulse sequence was used for data acquisition. The data were processed by inverse Laplace transformation (ILT), by using a maximum entropy algorithm, to afford 2D DOSY spectra. Different diffusion regimes for organic ligands in the bound and unbound states were successfully discriminated, more particularly in [D 3 ]acetonitrile, thus allowing assessment of their interactions with the nanoparticles.

Tripodal scaffold 1 has been used in the synthesis of a representative member of a library of serine protease mimics, possessing three independent functionalized peptide chains on a central core. Each peptide chain contains one residue of the classical catalytic triad (serine, histidine and aspartate) found in the active site of the serine protease α-chymotrypsin. A particular feature of the novel tripodal design is its essentially flexible yet preorganized structure as deduced from molecular modeling studies. The choice of suitable scaffold and side-chain protecting groups was intensively studied and optimized to ensure complete orthogonality. Inclusion of a photolabile linker enabled the release of intermediates and final structures from the solid-support polymer allowing positive evaluation of the present strategy for the synthesis of future tripodal libraries of serine protease mimics.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

We used trioctylphosphine oxide (TOPO) capped colloidal InP nanocrystals (Q-InP|TOPO) to explore the potential of solution 1H NMR spectroscopy in studying in situ the capping and capping exchange of sterically stabilized colloidal nanocrystals. The spectrum of Q-InP|TOPO shows resonances of free TOPO, superimposed on broadened spectral features. The latter were assigned to TOPO adsorbed at Q-InP by means of pulsed field gradient diffusion NMR and 1H-13C HSQC spectroscopy. The diffusion coefficient of Q-InP|TOPO nanocrystals was inferred from the decay of the adsorbed TOPO NMR signal. The corresponding hydrodynamic diameter correlates well with the diameter of Q-InP. By using the resolved methyl resonance of adsorbed TOPO, the packing density of TOPO at the InP surface can be estimated. Spectral hole burning was used to demonstrate explicitly that the adsorbed TOPO resonances are heterogeneously broadened. Exchange of the TOPO capping by pyridine was demonstrated by the disappearance of the resonances for adsorbed TOPO and the appearance of pyridine resonances in the 1H NMR spectrum. These results show that solution NMR spectroscopy should be considered a powerful technique for the in situ study of the capping of sterically stabilized colloidal nanocrystals.

Dialkyltin dichloride grafted to a cross-linked polystyrene, with the formula [P-H] ( 1 - 1 ) [P-( CH 2 ),,SnBuCL], (P= [CH 2 CH(pC 6 H 4 ), t= the degree of functionalization, and n=6 . or 11), is investigated as a recyclablecatalyst in the ring-opening polymerization (ROP) of e-caprolactone (CL). It is demonstrated that high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy is an invaluable tool to characterize completely the supported catalyst. The 2D 1 H- 1 3 C HSQC HR-MAS spectrum, in particular, allowed extensive assignment of the 1 H and 1 3 C resonances, as well as accurate measurement of the "J('H- 1 1 7 / 1 1 9 sn) and n J( 1 3 C- 1 1 7 / 1 1 9 Sn) coupling constants. 1 H and 1 1 9 Sn HR-MAS NMR spectroscopy is presented as a monitoring tool for catalytic processes based on organotin compounds, particularly for the investigation of the extent to which polymerization residues are observable in situ in the material pores and for the assessment of the chemical integrity and recycling conditions of the grafted catalyst. From polymerization experiments with CL. initiated by n-propanol and with [P-H] ( 1 - 1 ) ,[P-(CH 2 ) n SnBuCI 2 ], of various compositions as the supported catalyst, it appears that a partial 'burst' of the polystyrene support occurs when the length of the alkyl spacer is limited to n=-6, as a result of polymer chains growing within the pores of the support. However, extension of the length of the aliphatic polymethylene spacer from 6 to 11 carbon atoms preserves the support integrity and allows the production of catalyst-deprived poly(e-caprolactone) (PCL) oligomers. A preliminary attempt to recycle the heterogeneous catalyst has shown that very good reproducibility can be obtained, in terms of both catalyst activity and molecular-weight parameters of the asrecovered PCL polyester chains.

It is demonstrated that long-range nJ(1H,117Sn) coupling constants down to 0.3 Hz, can be accurately quantified from non-linear fitting of the sine modulation of the associated 1H117Sn correlation cross-peak intensities sampled as a function of the heteronuclear antiphase coherence preparation time in the 1H117Sn J-HMQC pulse sequence. The contribution of additional, undesired modulations is illustrated and assessed using the product operator formalism, and is traced back to contributions that arise from miss-setting of the wandering 180° pulse angle in the constant time period. The power of the method and its use in the characterization of weak intramolecular donor–acceptor interactions are illustrated by the determination of long-range nJ(1H,117Sn) coupling constants of bis[3-(dimethylamino)propyl]tin derivatives, [Me2N(CH2)3]2SnR2 (2, R = Me; 3, R = Ph; 4, R = t-Bu). By comparing these with the values found for the corresponding bis(4-methylpentyl)tin derivatives, [Me2CH(CH2)3]2SnR2 (2′, R = Me; 3′, R = Ph), which lack such interactions, the use of long-range coupling constants to detect intramolecular donor–acceptor interactions is evaluated. It is concluded that nJ(1H,117Sn) couplings up to six bonds through an organic carbon chain can be quantified, whether donor–acceptor interactions are present or not. Furthermore, evidence is presented that, when two scalar coupling pathways co-exist, the pathway involving an intramolecular donor–acceptor interaction can have opposite sign, thus decreasing the overall coupling constant to a value smaller than that actually measured in the absence of a donor–acceptor interaction, where only one coupling pathway is active. There is nevertheless clear numerical value discrimination in the series of compounds investigated between long-range couplings in derivatives without weak intramolecular donor–acceptor interactions and those where such interactions can exist. Copyright © 2004 John Wiley & Sons, Ltd.

Insoluble polystyrene-grafted compounds of the type [(P−H)(1−t){P−(CH2)6SnBuCl2}t] and [(P−H)(1−t)({P−(CH2)6SnBuX}2O)t/2], where (P−H) represents a cross-linked polystyrene, X is Cl, OH or OOCCH3, and t is the degree of functionalization, were synthesized from Amberlite XE-305, a polystyrene cross-linked with divinylbenzene. The compounds were characterized by elemental analysis, IR and Raman spectroscopy, solid-state 13C and 117Sn NMR spectroscopy and, where appropriate, by 119Sn hr-MAS NMR spectroscopy. The influence of the variable t and tin functionality on the catalytic activity of these compounds was assessed in the transesterification reaction of ethyl acetate with octanol. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

Headpiece (HP) is a 76-residue F-actin-binding module at the C terminus of many cytoskeletal proteins. Its 35-residue C-terminal subdomain is one of the smallest known motifs capable of autonomously adopting a stable, folded structure in the absence of any disulfide bridges, metal ligands, or unnatural amino acids. We report the three-dimensional solution structures of the C-terminal headpiece subdomains of human villin (HVcHP) and human advillin (HAcHP), determined by two-dimensional 1H-NMR. They represent the second and third structures of such C-terminal headpiece subdomains to be elucidated so far. A comparison with the structure of the chicken villin C-terminal subdomain reveals a high structural conservation. Both C-terminal subdomains bind specifically to F-actin. Mutagenesis is used to demonstrate the involvement of Trp 64 in the F-actin-binding surface. The latter residue is part of a conserved structural feature, in which the surface-exposed indole ring is stacked on the proline and lysine side chain embedded in a PXWK sequence motif. On the basis of the structural and mutational data concerning Trp 64 reported here, the results of a cysteine-scanning mutagenesis study of full headpiece, and a phage display mutational study of the 69–74 fragment, we propose a modification of the model, elaborated by Vardar and coworkers, for the binding of headpiece to F-actin.

Pulsed field gradient 1 H NMR spectroscopy has been applied to investigate the association behavior of the Sn 1 2 -oxo cluster macrocation [(BuSn) 1 2 O 1 4 (OH) 6 ] 2 + with two different and smaller anions, p-toluenesulfonate (PTS - ) and diphenylphosphinate (Ph 2 PO 2 -). By monitoring the translational diffusion coefficients of the various species involved, it is shown that the association depends on the anion involved and on the solvent used. Moreover, the possibility to individually monitor the diffusion characteristics of multiple anionic and cationic species in mixtures, by virtue of resolved 1 H resonances available from each species, allows us to evidence the occurrence of ion exchange in such systems. Thus when [(BuSn) 1 2 O 1 4 (OH) 6 ](PTS) 2 is mixed with two equivalents of Ph 2 PO 2 NMe 4 , PTS- is displaced by Ph 2 PO 2 -, highlighting the greater affinity of the organotin macrocation for the diphenylphosphinate. This example clearly illustrates the potential of pulsed field gradient 1 H NMR spectroscopy in inorganic/organometallic chemistry, to follow preferential ion pairing in multi-ion systems at the level of each individually charged species.

The selective recognition of the orthophosphate anion by a series of bifunctional Lewis acidic organotin compounds is investigated. The binding affinity of these carriers to anions, as measured by NMR titrations in CH2Cl2 obeyed the potentiometric selectivity order phosphate > sulfate > perchlorate, and provided the corresponding complex-formation constants. More accurate calculations of these values were obtained by the segmented-sandwich-membrane method performed directly inside the liquid polymeric membrane. These carriers were also studied potentiometrically in polymeric liquid membranes. The results indicate that (PhBr2Sn)2CH2 is 2 to 5 orders of magnitude more selective towards phosphate over other oxoanions. These results set the ground for the development of a new series of highly selective anions carriers with a wide range of possible applications.

The most abundant alpha-amylase inhibitor (AAI) present in the seeds of Amaranthus hypochondriacus, a variety of the Mexican crop plant amaranth, is the smallest polypeptide (32 residues) known to inhibit alpha-amylase activity of insect larvae while leaving that of mammals unaffected. In solution, 1H NMR reveals that AAI isolated from amaranth seeds adopts a major trans (70%) and minor cis (30%) conformation, resulting from slow cis-trans isomerization of the Val15-Pro16 peptide bond. Both solution structures have been determined using 2D 1H-NMR spectroscopy and XPLOR followed by restrained energy refinement in the consistent-valence force field. For the major isomer, a total of 563 distance restraints, including 55 medium-range and 173 long-range ones, were available from the NOESY spectra. This rather large number of constraints from a protein of such a small size results from a compact fold, imposed through three disulfide bridges arranged in a cysteine-knot motif. The structure of the minor cis isomer has also been determined using a smaller constraint set. It reveals a different backbone conformation in the Pro10-Pro20 segment, while preserving the overall global fold. The energy-refined ensemble of the major isomer, consisting of 20 low-energy conformers with an average backbone rmsd of 0.29 +/- 0.19 A and no violations larger than 0.4 A, represents a considerable improvement in precision over a previously reported and independently performed calculation on AAI obtained through solid-phase synthesis, which was determined with only half the number of medium-range and long-range restraints reported here, and featured the trans isomer only. The resulting differences in ensemble precision have been quantified locally and globally, indicating that, for regions of the backbone and a good fraction of the side chains, the conformation is better defined in the new solution structure. Structural comparison of the solution structure with the X-ray structure of the inhibitor when bound to its alpha-amylase target in Tenebrio molitor shows that the backbone conformation is only slightly adjusted on complexation, while that of the side chains involved in protein-protein contacts is similar to those present in solution. Therefore, the overall conformation of AAI appears to be predisposed to binding to its target alpha-amylase, confirming the view that it acts as a lid on top of the alpha-amylase active site.

The interactions between diethyltin dichloride and 5'-CMP, 5'-dCMP, and 5'-UMP in aqueous solution were investigated by multinuclear 1D and 2D NMR techniques including 119Sn, 15N and 31P nuclei. These studies were combined with electrospray mass spectrometry, infrared spectroscopy, solid state 13C, 31P and 117Sn CP-MAS NMR, and elemental analysis. As demonstrated by 31P-1H HOESY spectroscopy, the diethyltin moiety interacts with the phosphate group of the pyrimidic mononucleotides in the pH range 0.5-3.5. Compound 8 (X = Cl), the solid isolated in this pH range from 5'- CMP, contains two tin atoms bridged by one oxygen and one chlorine atom, each tin atom being linked to the phosphate group of a nucleotide moiety. For 5'- UMP the solid isolated (12) has a dimeric structure with two different tin atoms; it can be formed by dimerization of compound 11 with the elimination of two water molecules. As demonstrated by 1H-119Sn HMQC correlation NMR data and the 2J(119Sn-O-117Sn) coupling constant of 156 Hz, the diethyltin moiety reacts with the O(2') and O(3') oxygen atoms of the nucleotides to form a dimeric diethyldioxastannolane at pH > 9.0. Between pH 5.0 and 9.0, no evidence for any interaction between the diethyltin moiety and the nucleotides was found.

The selective recognition of the orthophosphate anion by a series of bifunctional Lewis acidic organotin compounds is investigated. The binding affinity of these carriers to anions, as measured by NMR titrations in CH2Cl2 obeyed the potentiometric selectivity order phosphate > sulfate > perchlorate, and provided the corresponding complex-formation constants. More accurate calculations of these values were obtained by the segmented-sandwich-membrane method performed directly inside the liquid polymeric membrane. These carriers were also studied potentiometrically in polymeric liquid membranes. The results indicate that (PhBr2Sn)2CH2 is 2 to 5 orders of magnitude more selective towards phosphate over other oxoanions. These results set the ground for the development of a new series of highly selective anions carriers with a wide range of possible applications.

The NMR conformational study of three asymmetric phenylindenylidene ruthenium complexes 4.1-4.3, is presented. Complete (1)H and (13)C assignments could be obtained for 4.1-4.3 in benzene solution from multiple 2D homonuclear and heteronuclear NMR techniques. Our NMR analysis shows that each complex exists as a 55:45 mixture of two rotational isomers in slow exchange on the NMR chemical shift timescale. They are shown to be related by a 180 degrees flip of the indenylidene ligand along the Ru=CR bond. Both rotational isomers can be discriminated by means of NOEs contacts between the various ligands coordinating to the Ru. By matching these stereospecific assignments to the chemical shift, a chemical shift based fingerprint of the isomers that may allow straightforward assignment of future asymmetric phenylindenylidene ruthenium complexes is proposed.

Because of its presence in many molecules of biological relevance, the conformational analysis of five-membered rings using (3)J(HH) scalar coupling data from NMR is a topic of considerable interest. Typically, conformational analysis involves the use of a well-established mathematical procedure, originally developed by de Leeuw et al., that fits two rigid conformations to the available experimental data. This so-called pseudorotation analysis approach is not without problems, however, as chemically unrealistic conformations are sometimes generated from the data. Here, we present our investigations in the use of time-averaged restrained molecular dynamics simulations as a generic tool to determine the conformations that agree with experimental (3)J(HH) scalar coupling data. For this purpose, a set of six ribose-based molecules has been used as model compounds. The influence of several modeling parameters is assessed and optimized values are proposed. The results obtained with the tar-MD approach arecompared to those obtained from the two conformer fitting procedure. Interpretation of the latter is facilitated by the introduction of a fitting error analysis that allows mapping the solution space of the fitting procedure. The relative merits of both methods and the advantages that result from the use of a force field and a time-averaged restraint potential for the experimental data are discussed. When combined, both techniques allow an enhanced understanding of the molecules' conformational behavior and prevent possible overinterpretation. In view of the very reasonable computational burden of a tar-MD Simulation for the systems investigated here, the approach should be generally applicable. (C) 2009 Wiley Periodicals, Inc. J Comput Chem 31: 561-572, 2010

1H, 117Sn J-HMBC spectroscopy was implemented as a tool for the determination of long range nJ(1H, 117Sn) coupling constants of [2-(N,N-dimethylaminomethyl)phenyl]stannanes, ArR1R2R3Sn or Ar2R1R2Sn, where R1, R2 and R3 are Ph or n-Bu, and/or Cl or Br. It is demonstrated that coupling constants which are too small to be measured from multiplet splittings in the 1H or 117Sn spectra can be measured from the development of the sine modulation of the intensity of the 1H, 117Sn HMBC correlation peaks as a function of the incremented preparation time of the 1H,117Sn HMBC pulse sequence. It is examined whether such couplings can be used as straightforward indicators for the existence of intramolecular nitrogen–tin interactions. While some coupling constants are shown to depend on the strength of the above-mentioned interaction, it is also demonstrated that 6J(1H,117Sn) coupling constants between the N-methyl groups and the tin nucleus can be observed even in structures where no nitrogen–tin interactions are possible. Copyright © 2001 John Wiley & Sons, Ltd.

The original article to which this Corrigendum refers was published in Magnetic Resonance in Chemistry 2004; 42: 776–780