NOE Interactions Research Articles

Study on the interactions between RS-licarbazepine and carboxyethylated cyclodextrins by electrokinetic chromatography and nuclear magnetic resonance

The interactions taking place in the enantiomeric separation of the antiepileptic drug licarbazepine by Electrokinetic Chromatography (EKC) using two carboxyethylated cyclodextrins (CE-CDs) (CE-β-CD and CE-γ-CD) as chiral selectors were investigated in this work to intend to explain the reversal in the enantiomer migration order observed depending on the CD. As these CDs were mixtures of isomers, only apparent and estimated association constants for enantiomer-CD complexes could be calculated based on EKC experiments, which agreed with the enantiomer migration order observed for CE-β-CD but not for CE-γ-CD. Additional experiments were carried out using NMR to study the stoichiometry of the complexes formed, their association constants and their structure. The construction of the corresponding Job plots, from 1H NMR titrations and intermolecular NOE interactions (between some of licarbazepine’s aromatic protons and the internal hydrogens of both CDs), permitted to conclude in the formation of 1:1 inclusion complex in both cases. Also, Scott plots for both systems were obtained showing a linear behavior for CE-β-CD while a non-linear behavior was observed for CE-γ-CD. As a result, only the apparent association constant for licarbazepine-CE-β-CD complexes was calculated (0.309 mM−1) (no significant differences between licarbazepine enantiomers were found in all NMR experiments). The structure of the complexes originated by licarbazepine and both CE-CDs was investigated. Insertion complexes were formed in each case, although some structural differences were noticed as the licarbazepine-CE-γ-CD complex would be less tight than that with CE-β-CD. Molecular dynamics (MD) simulations corroborated the structures derived from NMR results.

A lowly populated, transient β-sheet structure in monomeric Aβ1-42 identified by multinuclear NMR of chemical denaturation

Chemical denaturation is a well-established approach for probing the equilibrium between folded and unfolded states of proteins. We demonstrate applicability of this method to the detection of a small population of a transiently folded structural element in a system that is often considered to be intrinsically fully disordered. The 1HN, 15N, 13Cα, and 13C′ chemical shifts of Aβ1-40 and Aβ1-42 peptides and their M35-oxidized variants were monitored as a function of urea concentration and compared to analogous urea titrations of synthetic pentapeptides of homologous sequence. Fitting of the chemical shift titrations yields a 10 ± 1% population for a structured element at the C-terminus of Aβ1-42 that folds with a cooperativity of m = 0.06 kcal/mol·M. The fit also yields the chemical shifts of the folded state and, using a database search, for Aβ1-42 these shifts identified an antiparallel intramolecular β-sheet for residues I32-A42, linked by a type I′ β-turn at G37 and G38. The structure is destabilized by oxidation of M35. Paramagnetic relaxation rates and two previously reported weak, medium-range NOE interactions are consistent with this transient β-sheet. Introduction of the requisite A42C mutation and tagging with MTSL resulted in a small stabilization of this β-sheet. Chemical shift analysis suggests a C-terminal β-sheet may be present in Aβ1-40 too, but the turn type at G37 is not type I′. The approach to derive Transient Structure from chemical Denaturation by NMR (TSD-NMR), demonstrated here for Aβ peptides, provides a sensitive tool for identifying the presence of lowly populated, transiently ordered elements in proteins that are considered to be intrinsically disordered, and permits extraction of structural data for such elements.

Ancistrobrevines E-J and related naphthylisoquinoline alkaloids from the West African liana Ancistrocladus abbreviatus with inhibitory activities against Plasmodium falciparum and PANC-1 human pancreatic cancer cells

From the roots of the West African liana Ancistrocladus abbreviatus (Ancistrocladaceae), ten new naphthylisoquinoline alkaloids (7a, 7b, 8a, 8b, and 9–14), displaying three different coupling types (5,1′, 5,8′, and 7,8′), were isolated, among them a series of five 5,1′-linked representatives and four metabolites belonging to the rare group of 7,8′-coupled alkaloids. Two of the alkaloids, the ancistrobrevines I (13) and J (14), are only the fourth and fifth examples of 7,8′-linked naphthyldihydroisoquinolines ever found in nature. The stereostructures of the new plant metabolites were determined by spectroscopic, chemical (oxidative degradation), and chiroptical (electronic circular dichroism) methods. For the assignment of the axial configuration of 13 and 14 relative to the stereocenter at C-3, which is too far away for significant NOE long-range interactions, these 7,8′-coupled naphthyldihydroisoquinolines were stereoselectively converted into the respective cis-configured tetrahydroisoquinoline analogs. The newly generated ‘auxiliary’ stereocenter at C-1 permitted decisive NOE interactions between the isoquinoline and the naphthalene parts, and thus a reliable attribution of the axial configuration of 13 and 14. In addition, five known compounds (3, 5, 16, 17, and 20), previously discovered in related African and Asian Ancistrocladus species, have now for the first time been identified in A. abbreviatus. All of these alkaloids are S-configured at C-3 and bear an oxygen function at C-6, and are, thus, typical Ancistrocladaceae-type compounds. Some of the alkaloids of A. abbreviatus exhibited promising activities against the malaria parasite Plasmodium falciparum and PANC-1 human pancreatic cancer cells.

Four new diterpenes from the mangrove Ceriops tagal and structure revision of four dolabranes with a 4,18-epoxy group

Four new diterpenes named tagalons A-D (1–4), comprising an isopimarane (1), two 16-nor-pimaranes (2–3), and a dolabrane (4), were isolated from the Chinese mangrove, Ceriops tagal, together with four known dolabranes containing a 4,18-epoxy group, viz. tagalene I (5), 4-epitagalene I (6), tagalsin A (7), and tagalsin B (8). The structures of these compounds were unambiguously established by HR-ESIMS and NMR spectroscopic data. Based on the new criteria of NOE interactions between H2-18/H3-19 and H2-18/H3-20, previously reported relative configurations of the above four known dolabranes were correctly revised as their C-4 epimers, respectively. The relative configuration of tagalon B (2) and the absolute configuration of tagalene I (5) were established by single-crystal X-ray diffraction analyses, conducted with Cu Kα radiation. Most notably, tagalons C (3) and D (4) exhibited selective cytotoxicities against the human breast cancer cell line MT-1 with IC50 values of 3.75 and 8.07μM, respectively; whereas tagalene I (5) displayed potent cytotoxic effects against four human breast cancer cell lines MDA-MB-453, MDA-MB-231, SK-BR-3, and MT-1 with IC50 values of 8.97, 8.97, 4.62, and 3.93μM, respectively.

Structural and stereochemical elucidation of new hygrophorones from Hygrophorus abieticola (Basidiomycetes)

Four new hygrophorones (1–4) together with the known hygrophorone B12 (5) have been isolated from fruiting bodies of the basidiomycete Hygrophorus abieticola Krieglst. ex Gröger & Bresinsky. Their structures were assigned on the basis of extensive one and two dimensional NMR spectroscopic analyses as well as ESI-HRMS measurements. Among these compounds, two previously undescribed hygrophorone types, named hygrophorone H12 (3) and 2,3-dihydrohygrophorone H12 (4), were identified. The absolute configuration of hygrophorone E12 (2) is suggested based on quantum chemical CD calculations, while a semisynthetic approach in conjunction with computational studies and analysis of NOE interactions allowed the stereochemical assignment of compounds 3 and 4. Additionally, semisynthetic derivatives of hygrophorone B12 (5) were generated by acetylation of the hydroxyl groups. The biological activity of the natural and semisynthetic hygrophorones was evaluated against phytopathogenic organisms, revealing that the α,β-unsaturated carbonyl functionality is likely to be an essential structural feature. Hygrophorone B12 (5) was identified as the most active compound, acting against both ascomycetous fungi and oomycetes.

Host-guest interactions between cyclodextrins and surfactants with functional groups at the end of the hydrophobic tail

The aim of this work was to investigate the influence of the incorporation of substituents at the end of the hydrophobic tail on the binding of cationic surfactants to α-, β-, and γ-cyclodextrins. The equilibrium binding constants of the 1:1 inclusion complexes formed follow the trend K1(α-CD)>K1(β-CD)≫K1(γ-CD), which can be explained by considering the influence of the CD cavity volume on the host-guest interactions. From the comparison of the K1 values obtained for dodecyltriethylammonium bromide, DTEAB, to those estimated for the surfactants with the substituents, it was found that the incorporation of a phenoxy group at the end of the hydrocarbon tail does not affect K1, and the inclusion of a naphthoxy group has some influence on the association process, slightly diminishing K1. This makes evident the importance of the contribution of hydrophobic interactions to the binding, the length of the hydrophobic chain being the key factor determining K1. However, the presence of the aromatic rings does influence the location of the host and the guest in the inclusion complexes. The observed NOE interactions between the aromatic protons and the CD protons indicate that the aromatic rings are partially inserted within the host cavity, with the cyclodextrin remaining close to the aromatic rings, which could be partially intercalated in the host cavity. To the authors’ knowledge this is the first study on the association of cyclodextrins with monomeric surfactants incorporating substituents at the end of the hydrophobic tail.

Structure and Absolute Configuration of Pseudohygrophorones A(12) and B(12), Alkyl Cyclohexenone Derivatives from Hygrophorus abieticola (Basidiomycetes).

Pseudohygrophorones A(12) (1) and B(12) (2), the first naturally occurring alkyl cyclohexenones from a fungal source, and the recently reported hygrophorone B(12) (3) have been isolated from fruiting bodies of the basidiomycete Hygrophorus abieticola Krieglst. ex Gröger & Bresinsky. Their structures were assigned on the basis of extensive one- and two-dimensional NMR spectroscopic analysis as well as ESI-HRMS measurements. The absolute configuration of the three stereogenic centers in the diastereomeric compounds 1 and 2 was established with the aid of (3)JH,H and (4)JH,H coupling constants, NOE interactions, and conformational analysis in conjunction with quantum chemical CD calculations. It was concluded that pseudohygrophorone A(12) (1) is 4S,5S,6S configured, while pseudohygrophorone B(12) (2) was identified as the C-6 epimer of 1, corresponding to the absolute configuration 4S,5S,6R. In addition, the mass spectrometric fragmentation behavior of 1-3 obtained by the higher energy collisional dissociation method allows a clear distinction between the pseudohygrophorones (1 and 2) and hygrophorone B(12) (3). The isolated compounds 1-3 exhibited pronounced activity against phytopathogenic organisms.

Folding Patterns in a Family of Oligoamide Foldamers.

A series of small, unsymmetrical pyridine-2,6-dicarboxylamide oligoamide foldamers with varying lengths and substituents at the end groups were synthetized to study their conformational properties and folding patterns. The @-type folding pattern resembled the oxyanion-hole motifs of enzymes, but several alternative folding patterns could also be characterized. Computational studies revealed several alternative conformers of nearly equal stability. These folding patterns differed from each other in their intramolecular hydrogen-bonding patterns and aryl-aryl interactions. In the solid state, the foldamers adopted either the globular @-type fold or the more extended S-type conformers, which were very similar to those foldamers obtained computationally. In some cases, the same foldamer molecule could even crystallize into two different folding patterns, thus confirming that the different folding patterns are very close in energy in spite of their completely different shapes. Finally, the best match for the observed NOE interactions in the liquid state was a conformation that matched the computationally characterized helix-type fold.

ChemInform Abstract: Total Synthesis of Tricladins A and B and Identification of Their Absolute Configuration.

A concise synthesis of both (S)- and (R)-enantiomers of tricladins A and B from l-Boc alanine was achieved. The diastereomeric intermediates were separated by chiral column chromatography, and the absolute configuration of the 2-position was assigned by observed NOE interactions with the known stereogenic center at the 5-position. By comparison of all synthesized final enantiomers with the corresponding natural products, we concluded that the natural tricladins A and B must have the (R)-configuration.

Oligomers and peptidomimetics consisting of methyl 3-amino-2,3-dideoxyhexopyranosiduronic acids

In search of new foldamers, the synthesis of homo and heterooligomeric tetramers, consisting of methyl 3-amino-2,3-dideoxyhexopyranosiduronic acids, from the respective dimers, is presented. Both the dimers and tetramers were subjected to molecular dynamics simulations, based on NOE interactions. These show the preferred conformations of presented sugar amino acids (SAAs) oligomers. The CD spectra of these oligomers depend on the configuration of the C3 carbon atom bearing the amide chromophore. Next, the syntheses of two peptidomimetics with SAA incorporated into a peptide chain, are presented. A Leu-enkephalin mimetic was subjected to molecular dynamics simulations, which show how a SAA influences the geometry of a peptidomimetic. A Tat1 protein mimetic is demonstrated to display an inhibitory influence on the activity of a proteasome. Its structure is studied on a basis of the CD and FTIR spectra.

Total synthesis of tricladins A and B and identification of their absolute configuration.

A concise synthesis of both (S)- and (R)-enantiomers of tricladins A and B from l-Boc alanine was achieved. The diastereomeric intermediates were separated by chiral column chromatography, and the absolute configuration of the 2-position was assigned by observed NOE interactions with the known stereogenic center at the 5-position. By comparison of all synthesized final enantiomers with the corresponding natural products, we concluded that the natural tricladins A and B must have the (R)-configuration.

Isolation and Characterization of Two Geometric Allene Oxide Isomers Synthesized from 9S-Hydroperoxylinoleic Acid by Cytochrome P450 CYP74C3: STEREOCHEMICAL ASSIGNMENT OF NATURAL FATTY ACID ALLENE OXIDES

Specialized cytochromes P450 or catalase-related hemoproteins transform fatty acid hydroperoxides to allene oxides, highly reactive epoxides leading to cyclopentenones and other products. The stereochemistry of the natural allene oxides is incompletely defined, as are the structural features required for their cyclization. We investigated the transformation of 9S-hydroperoxylinoleic acid with the allene oxide synthase CYP74C3, a reported reaction that unexpectedly produces an allene oxide-derived cyclopentenone. Using biphasic reaction conditions at 0 °C, we isolated the initial products and separated two allene oxide isomers by HPLC at -15 °C. One matched previously described allene oxides in its UV spectrum (λmax 236 nm) and NMR spectrum (defining a 9,10-epoxy-octadec-10,12Z-dienoate). The second was a novel stereoisomer (UV λmax 239 nm) with distinctive NMR chemical shifts. Comparison of NOE interactions of the epoxy proton at C9 in the two allene oxides (and the equivalent NOE experiment in 12,13-epoxy allene oxides) allowed assignment at the isomeric C10 epoxy-ene carbon as Z in the new isomer and the E configuration in all previously characterized allene oxides. The novel 10Z isomer spontaneously formed a cis-cyclopentenone at room temperature in hexane. These results explain the origin of the cyclopentenone, provide insights into the mechanisms of allene oxide cyclization, and define the double bond geometry in naturally occurring allene oxides.

The interaction of nemorubicin metabolite PNU-159682 with DNA fragments d(CGTACG)2, d(CGATCG)2 and d(CGCGCG)2 shows a strong but reversible binding to G:C base pairs

The antitumor anthracycline nemorubicin is converted by human liver microsomes to a major metabolite, PNU-159682 (PNU), which was found to be much more potent than its parent drug toward cultured tumor cells and in vivo tumor models. The mechanism of action of nemorubicin appears different from other anthracyclines and until now is the object of studies. In fact PNU is deemed to play a dominant, but still unclear, role in the in vivo antitumor activity of nemorubicin. The interaction of PNU with the oligonucleotides d(CGTACG)2, d(CGATCG)2 and d(CGCGCG)2 was studied with a combined use of 1H and 31P NMR spectroscopy and by ESI-mass experiments. The NMR studies allowed to establish that the intercalation between the base pairs of the duplex leads to very stable complexes and at the same time to exclude the formation of covalent bonds. Melting experiments monitored by NMR, allowed to observe with high accuracy the behaviour of the imine protons with temperature, and the results showed that the re-annealing occurs after melting. The formation of reversible complexes was confirmed by HPLC–tandem mass spectra, also combined with endonuclease P1digestion. The MS/MS spectra showed the loss of neutral PNU before breaking the double helix, a behaviour typical of intercalators. After digestion with the enzyme, the spectra did not show any compound with PNU bound to the bases. The evidence of a reversible process appears from both proton and phosphorus NOESY spectra of PNU bound to d(CGTACG)2 and to d(CGATCG)2. The dissociation rate constants (koff) of the slow step of the intercalation process, measured by 31P NMR NOE-exchange experiments, showed that the kinetics of the process is slower for PNU than for doxorubicin and nemorubicin, leading to a 10- to 20-fold increase of the residence time of PNU into the intercalation sites, with respect to doxorubicin. A relevant number of NOE interactions allowed to derive a model of the complexes in solution from restrained MD calculations. The conformation of PNU bound to the oligonucleotides was also derived from the coupling constant values.

MTMDAT-HADDOCK: High-throughput, protein complex structure modeling based on limited proteolysis and mass spectrometry

BackgroundMTMDAT is a program designed to facilitate analysis of mass spectrometry data of proteins and biomolecular complexes that are probed structurally by limited proteolysis. This approach can provide information about stable fragments of multidomain proteins, yield tertiary and quaternary structure data, and help determine the origin of stability changes at the amino acid residue level. Here, we introduce a pipeline between MTMDAT and HADDOCK, that facilitates protein-protein complex structure probing in a high-throughput and highly automated fashion.ResultsA new feature of MTMDAT allows for the direct identification of residues that are involved in complex formation by comparing the mass spectra of bound and unbound proteins after proteolysis. If 3D structures of the unbound components are available, this data can be used to define restraints for data-driven docking to calculate a model of the complex. We describe here a new implementation of MTMDAT, which includes a pipeline to the data-driven docking program HADDOCK, thus streamlining the entire procedure. This addition, together with usability improvements in MTMDAT, enables high-throughput modeling of protein complexes from mass spectrometry data. The algorithm has been validated by using the protein-protein interaction between the ubiquitin-binding domain of proteasome component Rpn13 and ubiquitin. The resulting structural model, based on restraints extracted by MTMDAT from limited proteolysis and modeled by HADDOCK, was compared to the published NMR structure, which relied on twelve unambiguous intermolecular NOE interactions. The MTMDAT-HADDOCK structure was of similar quality to structures generated using only chemical shift perturbation data derived by NMR titration experiments.ConclusionsThe new MTMDAT-HADDOCK pipeline enables direct high-throughput modeling of protein complexes from mass spectrometry data. MTMDAT-HADDOCK can be downloaded from http://www.ifm.liu.se/chemistry/molbiotech/maria_sunnerhagens_group/mtmdat/together with the manual and example files. The program is free for academic/non-commercial purposes.

Determination of the Structures of Symmetric Protein Oligomers from NMR Chemical Shifts and Residual Dipolar Couplings

Symmetric protein dimers, trimers, and higher-order cyclic oligomers play key roles in many biological processes. However, structural studies of oligomeric systems by solution NMR can be difficult due to slow tumbling of the system and the difficulty in identifying NOE interactions across protein interfaces. Here, we present an automated method (RosettaOligomers) for determining the solution structures of oligomeric systems using only chemical shifts, sparse NOEs, and domain orientation restraints from residual dipolar couplings (RDCs) without a need for a previously determined structure of the monomeric subunit. The method integrates previously developed Rosetta protocols for solving the structures of monomeric proteins using sparse NMR data and for predicting the structures of both nonintertwined and intertwined symmetric oligomers. We illustrated the performance of the method using a benchmark set of nine protein dimers, one trimer, and one tetramer with available experimental data and various interface topologies. The final converged structures are found to be in good agreement with both experimental data and previously published high-resolution structures. The new approach is more readily applicable to large oligomeric systems than conventional structure-determination protocols, which often require a large number of NOEs, and will likely become increasingly relevant as more high-molecular weight systems are studied by NMR.

Solution Structure of a Novel Heterocyclic Dication Stacked as a Dimer with a Unique Mixed GC/AT Containing Motif

The search for small-molecules that can target regulatory nucleic acid elements is an ever-growing field driven by the passion that such molecules can potentially alter gene expression and thus influence diverse cellular functions. In recent years, small-molecules preferentially binding to G•C sequences have attracted the scientific community to further understand and establish DNA base-pair recognition rules. Biophysical studies from our group have shown that DB293, a phenyl-furan-benzimidazole aromatic dication, and DB1878, an indole analog, target G•C containing sequences in a highly cooperative manner. Current high resolution NMR studies of the two compounds with 5′-CTATGACTCTCGTCATAG-3′ hairpin sequence (binding site highlighted) have confirmed a strong cooperative dimerization of the compound at the 5′-ATGA-3′ site. Strong NOE interactions between DB1878 and specific DNA protons along the minor groove indicate an anti-parallel orientation of the two molecules in the minor-groove. Very interestingly, the two DB1878 molecules recognize the ATGA sequence in what we termed as an “in-out” model, where the furan rings of the two molecules are opposite to each other. This unique stacking mode enables the furan ring of one of the molecules to recognize the guanine amino group. Mutation studies show that replacing the central G•C base-pair with any other base completely altered the binding affinity, thereby suggesting the importance of that base-pair for compound recognition. Further analyses of the complex have shown that the two DB1878 molecules stack with optimal cation-pi type interactions and with favorable van der Waals interactions along the minor groove. The structure reported here is completely different from known DNA recognition, and represents an entirely new DNA minor groove recognition paradigm. Supported by NIAID Grant AI64200.

Structure Elucidation of Procyanidin Oligomers by Low-Temperature 1H NMR Spectroscopy

Procyanidin dimers and trimers, needed as reference compounds for biological studies, have been synthesized from various natural sources using a semisynthetic approach and purified by high-speed countercurrent chromatography (HSCCC). In the past, it has been difficult to elucidate the structure of these compounds, especially the determination of the interflavanoid bond. Here, the structure of two B-type procyanidin dimers, with (+)-catechin ((+)-C) in the upper unit, and eight C-type procyanidin trimers, with (-)-epicatechin ((-)-EC) in the upper unit, have been elucidated using low-temperature (1)H NMR spectroscopy, as well as circular dichroism (CD) spectroscopy. This is the first time NOE interactions have been used to characterize the interflavanoid linkage in underivatized procyanidin trimers. Complete analyses of procyanidin C1 (-)-EC-4β→8-(-)-EC-4β→8-(-)-EC, (-)-EC-4β→8-(-)-EC-4β→8-(+)-C, (-)-EC-4β→6-(-)-EC-4β→8-(-)-EC, (-)-EC-4β→6-(-)-EC-4β→8-(+)-C, (-)-EC-4β→8-(-)-EC-4β→6-(-)-EC, (-)-EC-4β→8-(-)-EC-4β→6-(+)-C, (-)-EC-4β→8-(+)-C-4α→8-(-)-EC, procyanidin C4 (-)-EC-4β→8-(+)-C-4α→8-(+)-C, and procyanidin dimers B6 (+)-C-4α→6-(+)-C and B8 (+)-C-4α→6-(-)-EC are presented.

A Cyclic [4]rotaxane that Behaves as a Switchable Molecular Receptor: Formation of a Rigid Scaffold from a Collapsed Structure by Complexation with Copper(I) Ions

The most efficient molecular receptors are usually rigid edifices with a hollow part that is able to accommodate the complexed species through an electronic and geometrical complementarity between the substrate and the complexing parts of the host. By analogy with biological processes related to induced fit, other host–guest processes are based on flexible hosts that are able to adapt their geometry to that of the species to be recognized. In the very active field of catenanes, rotaxanes, and molecular machines, very few systems have been considered as interesting receptors for molecular guests. One of the main contributions to this subfield of research is that of anion recognition by various interlocking compounds. Our research group has also recently described a [3]rotaxane that is able to act as an adjustable receptor. The system consists of two rings threaded by an axis on which the rings can move freely. The complexing units are zinc porphyrins that are firmly attached to the rotaxane rings and are able to interact with given substrates that consist of doubly end functionalized compounds bearing 4-pyridyl groups. It was shown that the marked geometrical adaptability of the host in its metal-free form allows interaction with guests of very different sizes. Herein we report the properties of a related compound, namely a cyclic bisporphyrin [4]rotaxane, the behavior of which is totally different from that of a previously studied linear [3]rotaxane. Contrary to the latter compound, the metal-free [4]rotaxane collapses completely and does not show any complexation properties, whereas the copper(I)complexed compound is a good and selective receptor for diamine and dipyridyl substrates because of the scaffolding effect of the four metal centers (Figure 1). The recognition process can thus be switched on and off by complexing the free ligand to four Cu ions or demetalating the metalcomplexed species, respectively. The synthesis of rotaxane 1 [7] as well as that of its related [4]pseudorotaxane have already been reported. 1 was demetalated using a large excess of KCN (ca. 50 equivalents) at room temperature. The reaction mixture was stirred for 2.5 hours, and the crude product was then purified by chromatography on silica gel to afford the demetalated rotaxane 2 in 88% yield (Scheme 1). Rotaxane 2 was characterized by H NMR spectroscopy (COSY, ROESY, DOSY), electrospray mass spectrometry and UV/Vis spectroscopy. Very surprisingly, the H NMR spectrum of rotaxane 2 shows considerable loss of symmetry compared to the metalated system 1, in which all the Cu centers as well as the four stoppers were chemically equivalent. A quarter of the rotaxane 1 only had thus to be considered for complete NMR assignment. By contrast, the H NMR signals of 2 were doubled compared to those of rotaxane 1. Two distinct stoppers with significantly different H NMR signals and thus markedly different environments can be identified. NOE interactions between some parts of 2 were clearly detected, whereas these fragments were too far away from one another in 1 to show any interaction. For instance, some protons of the tBu groups of one type of stopper (H-re’; r= “rod”) correlate to protons H-b4’ and Hb7’ (b=bismacrocycle) of the 1,10-phenanthroline unit in 2, thus indicating that this stopper and a given 1,10-phenanthroline unit are located very close to one another. As the bismacrocycles and the axles are both rigid, the proximity between two such fragments tends to indicate that the topography of 2 is markedly different from that of 1, and that 2 has a totally collapsed structure. This hypothesis was confirmed by additional observations. For instance, one proton of the (CH2)3 linker (H-r9) is strongly shielded upon decomplexation; the corresponding signal moves from Figure 1. Principle of the recognition process that is switched on and off by metalation or demetalation. Small gray dots: Cu ions; gray squares: porphyrins; black double arrow: guest compound. The chemical structures of 1 and 2 are shown in Scheme 1.

Three‐dimensional structure of the weakly associated protein homodimer SeR13 using RDCs and paramagnetic surface mapping

The traditional NMR-based method for determining oligomeric protein structure usually involves distinguishing and assigning intra- and intersubunit NOEs. This task becomes challenging when determining symmetric homo-dimer structures because NOE cross-peaks from a given pair of protons occur at the same position whether intra- or intersubunit in origin. While there are isotope-filtering strategies for distinguishing intra from intermolecular NOE interactions in these cases, they are laborious and often prove ineffectual in cases of weak dimers, where observation of intermolecular NOEs is rare. Here, we present an efficient procedure for weak dimer structure determination based on residual dipolar couplings (RDCs), chemical shift changes upon dilution, and paramagnetic surface perturbations. This procedure is applied to the Northeast Structural Genomics Consortium protein target, SeR13, a negatively charged Staphylococcus epidermidis dimeric protein (K(d) 3.4 ± 1.4 mM) composed of 86 amino acids. A structure determination for the monomeric form using traditional NMR methods is presented, followed by a dimer structure determination using docking under orientation constraints from RDCs data, and scoring under residue pair potentials and shape-based predictions of RDCs. Validation using paramagnetic surface perturbation and chemical shift perturbation data acquired on sample dilution is also presented. The general utility of the dimer structure determination procedure and the possible relevance of SeR13 dimer formation are discussed.

Interaction between double helix DNA fragments and the new antitumor agent sabarubicin, Men10755

Among the disaccharide derivatives of the antitumor anthracycline doxorubicin, sabarubicin (Men10755) is more active and less cytotoxic than doxorubicin. It showed a strong in vivo antitumor activity in all preclinical models examined, in conjunction with a better tolerability, and is now in phase II clinical trials.The interaction of sabarubicin and Men10749 (a similar disaccharide with a different configuration at C-4′ of the proximal sugar) with the hexanucleotides d(CGTACG)2 and d(CGATCG)2 was studied by a combined use of 2D-1H and 31P NMR techniques. Both 1H and 31P chemical shifts of imino protons and phosphates allowed to established the intercalation sites between the CG base pairs, as it occurs for other anthracyclines of the series. The dissociation rate constants (koff) of the slow step of the intercalation process were measured for Men10755 and Men10749, by NMR NOE-exchange experiments. The increase of koff , with respect of doxorubicin, showed that the intercalation process is significantly faster for both drugs, leading to an average residence time for sabarubicin into d(CGTACG)2 sixfold shorter than for doxorubicin. This could give account of both higher cytoplasmic/nuclear ratio and lower cellular uptake of sabarubicin in comparison with doxorubicin and accordingly of the lower cytotoxicity of these disaccharide analogues.A relevant number of NOE interactions allowed the structure of the complexes in solution to be derived through restrained MD calculations. NMR-DOSY experiments were performed with several drug/oligonucleotide mixtures in order to determine the structure and the dimension of the aggregates.