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In reactions between the hexamolybdate [Mo6O19I2-and 1,4-diaminobenzener terminal oxides are replaced by 4-aminophenylimido ligands and the first structurally characterised polyoxometallate to contain functionalised organoimido ligands, ( B U ~~N ) ~[ M O ~O ~~( N C ~H ~N H ~) ~I 5, is also the first example of a trans-bis(imido) derivative; further condensation also occurs to yield species in which hexamolybdate units are linked by Ir4-phenylenediimido bridges.

We report on the mobilization of shuttle plasmids from gram-negative Escherichia coli to gram-positive corynebacteria mediated by P-type transfer functions. Introduction of plasmids into corynebacteria was markedly enhanced after heat treatment of the recipient cells. High-frequency plasmid transfer was also observed when the restriction system of the recipient was mutated. On the basis of our data, we conclude that efficient DNA transfer from gram-negative to gram-positive bacteria, at least to coryneform bacteria, is conceivable in certain natural ecosystems.Conjugal transfer of broad-host-range IncP-type resistance plasmids within gram-negative bacterial species is well known (3, 9, 18). Non-self-transmissible plasmids carrying the appropriate origin of transfer (oriT) can be mobilized by IncP plasmids (28). Recent studies have shown that conjugation is a nonspecific process and accounts for most horizontal gene transfer between even phylogenetically remote organisms (7,13,25,26).To investigate the possibility of conjugal plasmid transfer between Escherichia coli and Corynebacterium glutamicum, we took advantage of a mobilization system previously developed for genetic engineering of a wide range of gramnegative bacteria (23). This strategy is based on the oriT and the transfer (Tra) functions of IncP-type broad-host-range plasmid RP4 (10) and consists of E. coli mobilizing strains and derivatives of conventional E. coli vectors (pSUP vectors [22,23]). A series of E. coli-C. glutamicum shuttle plasmids was constructed based on mobilizable E. coli vector pSUP102 (22). The 10.6-kilobase prototype shuttle vector pECM1 resulted from fusion of pSUP102 to C. glutamicum vector pCV35 (Fig. 1).Transfer of pECMl to C. glutamicum. For mating experiments, plasmid pECM1 was introduced by transformation into mobilizing strain E. coli S17-1 (23). E. coli S17-1 carries an RP4 derivative integrated into the chromosome which provides the transfer functions necessary for mobilization. By using this donor, plasmid pECM1 was transferred by conjugation as previously described (23) to a nalidixic acidresistant derivative of E. coli MM294 (14) at frequencies between 10-1 and 10-2 (Table 1) per donor cell. For conjugal transfer of pECM1 to coryneform recipient strains, donor strain S17-1(pECM1) was grown to the late-exponential phase in LB medium (15) containing 50 ,ug of kanamycin per ml. Recipient strains were grown in LB medium to an optical density at 580 nm of 3 to 4. About 7 x 108 donor and 3.5 x 109 recipient cells, corresponding to a ratio of 1:5, were mixed and pelleted by centrifugation at 20°C for a short time. The mating mixture was then carefully suspended in about 500 ,ll of LB medium and spread onto a 0.45-pum-pore-size cellulose acetate filter (Millipore Corp., Bedford, Mass.) placed on a prewarmed LB plate. After 20 h of incubation at 30°C, the cells were washed from the filter with 1 ml of LB medium and mechanical agitation. Transconjugants were * Corresponding author.

Patterned monolayers of N-heterocyclic carbenes (NHCs) on gold surfaces were obtained by microcontact printing of NHC-CO adducts and NHC(H)[HCO ] salts. The NHC-modified areas showed an increased conductivity compared to unmodified gold surface areas. Furthermore, the remaining surface areas could be modified with a second, azide-functionalized carbene, facilitating further applications and post-printing modifications. Thorough elucidation by a variety of analytical methods offers comprehensive evidence for the viability of the methodology reported here. The protocol enables facile access to versatile, microstructured NHC-modified gold surfaces with highly stable patterns, enhanced conductivity, and the option for further modification.

The epidermal growth factor receptor (EGFR) is upregulated within a high percentage of solid tumors and hence is an attractive target for tumor-targeted therapies including gene therapy. The natural EGFR ligand epidermal growth factor (EGF) has been used for this purpose, despite the risk of mitogenic effects due to EGFR activation. We have developed a fully synthetic, EGFR-targeted gene delivery system based on PEGylated linear polyethylenimine (LPEI), allowing evaluation of different EGFR-binding peptides in terms of transfection efficiency and EGFR activation. Peptide sequences directly derived from the human EGF molecule enhanced transfection efficiency with concomitant EGFR activation. Only the EGFR-binding peptide GE11, which has been identified by phage display technique, showed specific enhancement of transfection on EGFR-overexpressing tumor cells including glioblastoma and hepatoma, but without EGFR activation. EGFR targeting led to high levels of cell association of fluorescently labeled polyplexes after only 30 min of incubation. EGF pretreatment of cells induced enhanced cellular internalization of all polyplex types tested, pointing at generally enhanced macropinocytosis. EGF polyplexes diminished cell surface expression of EGFR for up to 4 hr, whereas GE11 polyplexes did not. In a clinically relevant orthotopic prostate cancer model, intratumorally injected GE11 polyplexes were superior in inducing transgene expression when compared with untargeted polyplexes.

Homo- and heterodivalent crown-ammonium pseudorotaxanes with different spacers connecting the two axle ammonium binding sites have been synthesized and characterized by NMR spectroscopy and ESI mass spectrometry. The homodivalent pseudorotaxanes are investigated with respect to the thermodynamics of divalent binding and to chelate cooperativity. The shortest spacer exhibits a chelate cooperativity much stronger than that of the longer spacers. On the basis of crystal structure, this can be explained by a noninnocent spacer, which contributes to the binding strength in addition to the two binding sites. Already very subtle changes in the spacer length, i.e., the introduction of an additional methylene group, cause substantial changes in the magnitude of cooperative binding as expressed in the large differences in effective molarity. With a similar series of heterodivalent pseudorotaxanes, the spacer effects on the barrier for the intramolecular threading step has been examined with the result that the shortest spacer causes a strained transition structure and thus the second binding event occurs slower than that of the longer spacers. The activation enthalpies and entropies show clear trends. While the longer spacers reduce the enthalpic strain that is present in the transition state for the shortest member of the series, the longer spacers become entropically slightly more unfavorable because of conformational fixation of the spacer chain during the second binding event. These results clearly show the noninnocent spacers to complicate the analysis of multivalent binding. An approximate description which considers the binding sites to be connected just by a flexible chain turns out to be more a rough approximation than a good model. The second conclusion from the results presented here is that multivalency is expressed in both the thermodynamics and the kinetics in different ways. A spacer optimized for strong binding is suboptimal for fast pseudorotaxane formation.

Three binary pseudorotaxanes, which are based on two different secondary ammonium/crown ether binding motifs, have been studied by (1)H NMR and (1)H,(1)H EXSY NMR experiments with respect to their thermodynamic stabilities and their axle exchange kinetics. The stability ranking does not follow the order of axle exchange rates, and the thermodynamically most stable axle-wheel combinations assemble only slowly. On the basis of these binding motifs, a series of self-sorting systems have been studied ranging from simple four-component mixtures through sequence-specific pseudorotaxanes to multiply threaded complexes. Because of the mismatch of kinetic and thermodynamic order, wrongly assembled structures are unavoidable, which require error-correction steps to yield the final thermodynamically controlled self-sorted products. These error-correction steps can easily be monitored by electrospray mass spectrometry, when a mixed-flow microreactor is coupled to the ion source to cover second time scales. Self-assembly intermediates, wrongly assembled structures, and the final thermodynamic products can be simultaneously identified. The determination of preferred assembly pathways as well as the formation of dead-end structures provides a clear picture of a rich kinetic behavior of the self-sorting systems under study.