Kathrin Meindl

A comparison of a microfocus X-ray source and a conventional sealed tube for crystal structure determination

Experiments are described in which a direct comparison was made between a conventional 2 kW water-cooled sealed-tube X-ray source and a 30 W air-cooled microfocus source with focusing multilayer optics, using the same goniometer, detector, radiation (Mo Kα), crystals and software. The beam characteristics of the two sources were analyzed and the quality of the resulting data sets compared. The Incoatec Microfocus Source (IµS) gave a narrow approximately Gaussian-shaped primary beam profile, whereas the Bruker AXS sealed-tube source, equipped with a graphite monochromator and a monocapillary collimator, had a broader beam with an approximate intensity plateau. Both sources were mounted on the same Bruker D8 goniometer with a SMART APEX II CCD detector and Bruker Kryoflex low-temperature device. Switching between sources simply required changing the software zero setting of the 2θ circle and could be performed in a few minutes, so it was possible to use the same crystal for both sources without changing its temperature or orientation. A representative cross section of compounds (organic, organometallic and salt) with and without heavy atoms was investigated. For each compound, two data sets, one from a small and one from a large crystal, were collected using each source. In another experiment, the data quality was compared for crystals of the same compound that had been chosen so that they had dimensions similar to the width of the beam. The data were processed and the structures refined using standard Bruker and SHELX software. The experiments show that the IµS gives superior data for small crystals whereas the diffracted intensities were comparable for the large crystals. Appropriate scaling is particularly important for the IµS data.

Labyrinthopeptins: A New Class of Carbacyclic Lantibiotics

Lantibiotics are peptides that are ribosomally synthesized from bacteria such as staphylococci, lactobacilli, and actinomycetes. The common structural characteristic of lantibiotics is the noncanonical amino acid lanthionine (Lan, 1; Figure 1), which confers conformational stability to the peptide. The most prominent representative is nisin, which is a lipid II binder, and has been known for its use as an antimicrobial food preservative for over 40 years. The majority of studies on molecular targets and bioactivities are focused on potential applications of lantibiotics as anti-infectives. Duramycin (Moli1901) is in phase II clinical trials for the treatment of cystic fibrosis because of its ability to increase chloride transport in airway epithelium. Biosurfactant function in the life cycle of streptomycetes has been elucidated for some members such as SapB. Herein, we present the structures, the biosynthesis gene cluster, and the bioactivities of labyrinthopeptins, which are lantibiotics that contain labionin, an unprecedented carbacyclic, posttranslationally modified amino acid. The culture extracts of the novel actinomycete Actinomadura namibiensis DSM 6313 attracted our attention because of their activity against the Herpes simplex virus. Active fractions of the extracts contained a peptide that was isolated by chromatographic methods. The high-resolution ESI-FTICR mass spectrum showed a mass of 984.3333 Da for the doubly charged sodium adduct of the compound, corresponding to a neutral monoisotopic mass of 1922.6872 Da and the molecular formula C85H110N20O24S4 (Dm/m= 0.7 ppm). Amino acid analysis revealed Gly and the l-enantiomers of Ala, Thr, Leu, Asx, Cys, Phe, Glx, Trp (ratio 1:1:1:2:1:2:1:1:2). However, the total molecular mass of the detected amino acids indicated a considerable mass difference, which could not be correlated with known peptidic or lantibiotic posttranslational modifications. Resolution of the structure by H NMR spectroscopy was impeded by broad signals in parts of the spectrum. The X-ray structure at 1.0 resolution (Figure 1) enabled interpretation of the analytical data and displayed several unique structural features. In view of its labyrinthine structure, the compound was named labyrinthopeptin A2 (2). Labyrinthopeptin A2 has a globular structure that consists primarily of hydrophobic amino acids. Formally, the structure can be dissected into two nonapeptides. Each peptide bears a C-terminal Cys residue that forms a disulfide bond, which is a comparatively rare modification in lantibiotics, but is found for sublancin 168 from B. subtilis. Each nonapeptide contains a tetrapeptide (ring A) and a pentapeptide (ring B) that share a quaternary aC atom; labyrinthopeptin A rings are formed by a methylene group between the aC atoms of Lab1/ Lab10 and Lab4/Lab13 (Figure 1). A carbacyclic side-chain linkage is unprecedented in peptides and proteins. We propose the name labionin (Lab) for the corresponding amino acid (Figure 1). Labionin 3 represents an aC quaternary substituted amino acid with a subtle structural resemblance to a-aminoisobutyric acid (Aib) or isovaline (Iva), which are incorporated in fungal peptaibol-type antibiotics. The stereocenters of 3 can be assigned to (2S,4S,8R)-labionin (Lab), which is consistent with the configuration of (2S,6R)lanthionine of other lantibiotics. The formation of the 11membered ring that involves 3 forces the peptide backbone into a conformation with cis-amide bonds between Asp2– Trp3 and Thr11–Gly12, respectively (Figure 1). The presence of cis-amide bonds and the absence of a hydrogen bond between Lab1–Lab4 and Lab10–Lab13, respectively, show that the turn motif in 2 is clearly different from a b-turn motif. Subsequent identification of the biosynthetic gene cluster was performed from a cosmid library of A. namibiensis by means of degenerated primer probes, followed by sequencing [*] Dr. T. Schmiederer, Dr. K. Schneider, Dr. A. Reicke, Dr. D. Butz, Dr. S. Keller, Prof. Dr. R. D. S ssmuth Technische Universit t Berlin, Fakult t II—Institut f r Chemie Strasse des 17. Juni 124, 10623 Berlin (Germany) Fax: (+49)30-314-24205 E-mail: suessmuth@chem.tu-berlin.de Homepage: http://www2.tu-berlin.de/fb5/Suessmuth/ contact.html Dr. K. Meindl, Prof. Dr. G. M. Sheldrick Universit t G ttingen (Germany)

Foundations of residual-density analysis

New and concise descriptors of the residual density are presented, namely the gross residual electrons, the net residual electrons and the fractal dimension distribution. These descriptors indicate how much residual density is present and in what way it is distributed, i.e. the extent to which the distribution is featureless. The amount of residual density present accounts for noise in the experimental data as well as for modeling inadequacies. Therefore, the minimization of the gross residual electrons during refinement serves as a quality criterion. In the case where only Gaussian noise is present in the residual density, the fractal distribution is parabolic in shape. Deviations from this shape therefore serve as an indicator for systematic errors. The new measures have been applied to simulated and experimental data in order to study the effects of noise, model inadequacies and truncation in the experimental resolution. These measures, although designed and examined with particular regard to applications of space residual density, are very general and can in principle also be applied to space and momentum residual densities in a one-, two-, three- or higher-dimensional Euclidean space.

Lewis-Base-Stabilized Dichlorosilylene: A Two-Electron σ-Donor Ligand

The first structurally described cobalt(I) Lewis-base-stabilized silylene complex [Co(CO)(3){SiCl(2)(IPr)}(2)](+)[CoCl(3)(THF)](-) [1; IPr = 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene] was prepared by applying the two-electron sigma-donor ligand SiCl(2)(IPr) through coordination with Co(2)(CO)(8). The bonding situation between ligand SiCl(2)(IPr) and the cobalt(I) metal center in [Co(CO)(3){SiCl(2)(IPr)}(2)](+) of 1 was investigated by (1)H NMR and IR spectroscopy, single-crystal X-ray structural analysis, and density functional theoretical calculations.

Exploiting tertiary structure through local folds for crystallographic phasing.

We describe an algorithm for phasing protein crystal X-ray diffraction data that identifies, retrieves, refines and exploits general tertiary structural information from small fragments available in the Protein Data Bank. The algorithm successfully phased, through unspecific molecular replacement combined with density modification, all-helical, mixed alpha-beta, and all-beta protein structures. The method is available as a software implementation: Borges.

Structure solution with ARCIMBOLDO using fragments derived from distant homology models

Molecular replacement, one of the general methods used to solve the crystallographic phase problem, relies on the availability of suitable models for placement in the unit cell of the unknown structure in order to provide initial phases. ARCIMBOLDO, originally conceived for ab initio phasing, operates at the limit of this approach, using small, very accurate fragments such as polyalanine α‐helices. A distant homolog may contain accurate building blocks, but it may not be evident which sub‐structure is the most suitable purely from the degree of conservation. Trying out all alternative possibilities in a systematic way is computationally expensive, even if effective. In the present study, the solution of the previously unknown structure of MltE, an outer membrane‐anchored endolytic peptidoglycan lytic transglycosylase from Escherichia coli, is described. The asymmetric unit contains a dimer of this 194 amino acid protein. The closest available homolog was the catalytic domain of Slt70 (PDB code 1QTE). Originally, this template was used omitting contiguous spans of aminoacids and setting as many ARCIMBOLDO runs as models, each aiming to locate two copies sequentially with PHASER. Fragment trimming against the correlation coefficient prior to expansion through density modification and autotracing in SHELXE was essential. Analysis of the figures of merit led to the strategy to optimize the search model against the experimental data now implemented within ARCIMBOLDO‐SHREDDER (http://chango.ibmb.csic.es/SHREDDER). In this strategy, the initial template is systematically shredded, and fragments are scored against each unique solution of the rotation function. Results are combined into a score per residue and the template is trimmed accordingly.

On the effect of neglecting anharmonic nuclear motion in charge density studies.

The effect of neglecting anharmonic nuclear motion when it is definitely present is studied. To ensure the presence of anharmonic nuclear motion a model was used that was previously refined against experimental data including anharmonic nuclear motion, and these calculated structure factors were used as observed data for a multipole refinement. It was then studied how the neglect of anharmonic nuclear motion and noise in the data affects the usual crystallographic quality measure R, the density parameters and the residual density distribution. It is demonstrated that the neglect of anharmonic nuclear motion leads to a characteristic imprint onto the residual density distribution in terms of residual density peaks and holes, in terms of the whole residual density distribution and in terms of the number, location and strength of valence shell charge concentrations (VSCCs). These VSCCs differ from that of the input model in a way which heavily influences and misleads the chemical interpretation of the charge density. This imprint vanishes after taking anharmonic nuclear motion into account. Also the input model VSCCs are restored. The importance of modeling anharmonic nuclear motion is furthermore shown by the characteristic imprint on the residual density distribution, even in the case of a numerically almost unaffected R value.

Experimental charge density studies of disordered N-phenylpyrrole and N-(4-fluorophenyl)pyrrole.

The static electron densities of the title compounds were extracted from high-resolution X-ray diffraction data using the nucleus-centered finite multipole expansion technique. The interpretation of the data collected for the N-phenylpyrrole crystal revealed a static disorder that could be successfully resolved within the aspherical-atom formalism. The local and integrated topological properties of the density obtained via a constrained multipole refinement are in statistical agreement with those calculated at the B3LYP/cc-pVTZ level of theory for the isolated molecule and for those derived from the experimental density of the para-fluorinated derivative N-(4-fluorophenyl)pyrrole. The topological analysis of the densities indicates neither pyramidal character of the pyrrole N-atom nor a quinoidal structure of the phenyl rings in either molecule. The fluorine substitution appears to have only a minor effect on the density of the remaining constituents but it results in markedly different features of the electrostatic potential of the two compounds. The consistency of the multipole refinement is validated by residual density analysis.

Synthesis and characterization of alkynyl complexes of groups 1 and 2.

The synthesis of N-heterocyclic carbene adducts of alkynyl lithium and magnesium is achieved, and different degrees of association are observed. Reaction of strontium amide nacnacSrN(SiMe(3))(2)(thf) (nacnac = CH(CMe2,6-iPr(2)C(6)H(3)N)(2)) with PhC[triple bond]CH in THF yields the dimeric alkynyl complex [nacnacSr(thf)(mu-C[triple bond]CPh)](2) which shows an interesting coordination geometry around the metal center. The compound retains the THF molecules, unlike its lighter congener, even in hydrocarbon solvents.

Consecutive donor-base exchange in anthracenyllithium compounds.

Organolithium compounds are of outstanding importance as starting materials for numerous products in synthetic chemistry and also in industrial processes. Their remarkably wide range of applications spans from deprotonation of weakly acidic compounds to transfer reactions of organic groups or even anionic polymerization reactions. Since Schlenk and Holtz reported the first syntheses of organolithium compounds, and the first crystal structure of a compound from this class (tetrameric ethyllithium) was successfully determined by Dietrich in 1963, a large number of alkyl and aryl lithium reagents has been structurally characterized. In hydrocarbon solvents, organolithium reagents form oligomers and their size significantly influences the reactivity. For the two compounds most commonly used in synthesis, nBuLi and tBuLi, the degree of oligomerization in solution was identified early on by cryoscopical and spectroscopical measurements. It was found that nBuLi forms a hexamer whereas tBuLi forms a tetramer in non-donating solvents. By addition of ethers, such as diethyl ether or THF, and especially by addition of tertiary amine donor bases, such as TMEDA or PMDETA, these oligomers can be disaggregated to smaller fragments, resulting in an enhanced reactivity. This is illustrated by the benzylic deprotonation of toluene employing nBuLi, which is only feasible upon the addition of TMEDA. In practice, mixtures of different donor bases are frequently used; however, the exact percentage of the single donor bases to give the most suitable complex is usually determined empirically, and the exact constitution of the reactive complex is unknown. It is most commonly presumed that the entire coordination sphere of the lithium atom is occupied by the strongest donor molecules available. Structural evidence for complexes in which organolithium compounds are coordinated by two different donor bases simultaneously are rare and their reproducibility is difficult. Using sterically demanding organolithium compounds, we embarked on monitoring the selective and consecutive donor base exchange. Herein, the complexes [R(C14H8)Li·{Et2O}n· {THF}m]2 were isolated and structurally characterized, and the lengths of the Li C bonds of 1a (n = 1, m = 0, R = Br), 2b (n = 1, m = 1/2, R = Me), 3 a (n = 1, m = 1, R = Br), 4c (n = 1/2, m = 3/2, R = Cl), and 5a (n = 0, m = 2, R = Br) are monitored, because they provide information on the polarity of the bonds and thus shed some light on the expected reactivities of the formed complexes. The degree of oligomerization, which mainly depends on the donor-base capacity, is additionally influenced by the steric demand of the alkyl or aryl carbanion, and is decreased with increasing size; for example, nBuLi is a hexamer whereas tBuLi forms a tetramer. By coordination of multidentate donor bases, the size of these aggregates can further be decreased, even to monomers: for example, [PhLi·Et2O]4 > [PhLi·TMEDA]2 > [PhLi·PMDETA]. Upon combination of weak monodentate donor bases such as diethyl ether with organolithium compounds of substantial steric bulk, small, mostly dimeric aggregates are formed. In these dimers, the lithium atoms are sterically shielded in a way that the coordination sphere can not be filled entirely to the preferred coordination number of four. Systems such as these are particularly suitable for monitoring the process of donor base uptake and exchange. In the following investigation we selected anthracene, which can formally be described as a diortho-substituted benzene. It is suitable as a proof-of-concept to facilitate the comparison to diortho-substituted phenyllithium derivatives omnipresent in the literature. The brominated anthracene derivatives can easily be obtained by the reaction of the monosubstituted anthracenes with elemental bromine in solution. Reacting nBuLi with bromoanthracenes gives the lithiated species in almost quantative yields (Scheme 1).