Application of ODMR to the study of DNA complexes of bisintercalating antibiotics and their biosynthesized derivatives

The DNA complexes of triostin A, echinomycin, and the monoquinoline (1QN) and bisquinoline (2QN) biosynthesized derivatives of echinomycin were investigated by optical detection of triplet-state magnetic resonance (ODMR) spectroscopy, with the quinoxaline and quinoline moieties of the DNA-binding peptides used as intrinsic probes. Plots of zero-field splitting (zfs)D parameter versus monitored wavelength revealed heterogeneity in the phosphorescence emission of echinomycin, triostin A, and 2QN ascribed to the occurrence of major and minor forms of the peptides in aqueous solution. ODMR results, in conjunction with findings from phosphorescence studies, indicate that the quinoxaline and quinoline chromophores of the major forms of the peptides are involved in aromatic stacking interactions in complexes with the natural DNAs fromMicrococcus lysodeikticus, Escherichia coli, and calf thymus as evidenced by red shifts in the phosphorescence 0,0 bands of the drugs, reductions in the phosphorescence lifetimes and zfsD andE parameters, and polarity reversal of the ODMR slow passage signals upon drug complexation. The reversal in ODMR signal polarity of echinomycin and 2QN is a consequence of changes in the triplet-state sublevel decay constants upon peptide binding to the natural DNAs. The extent of reduction of theD parameter for the major form of echinomycin, 2QN, and the quinoline moiety of 1QN upon complexation with polymeric DNAs was found to correlate with the binding affinities measured for these targets [1], but no correlation was found for the quinoxaline moiety of 1QN. Preliminary studies of triostin A-DNA complexes also revealed no correlation between the reduction in zfsD-value upon complexation and binding affinity, although the largest reductions inD-value among the peptides investigated in this report were exhibited by the poly(dG-dC)·poly(dG-dC) and natural DNA complexes of triostin A.     

Intermolecular packing and alignment in an ordered beta-hairpin antimicrobial peptide aggregate from 2D solid-state NMR

The aggregation and packing of a membrane-disruptive beta-hairpin antimicrobial peptide, protegrin-1 (PG-1), in the solid state are investigated to understand its oligomerization and hydrogen-bonding propensity. Incubation of PG-1 in phosphate buffer saline produced well-ordered nanometer-scale aggregates, as indicated by 13C and 15N NMR line widths, chemical shifts, and electron microscopy. Two-dimensional 13C and 1H spin diffusion experiments using C-terminus strand and N-terminus strand labeled peptides indicate that the beta-hairpin molecules in these ordered aggregates are oriented parallel to each other with like strands lining the intermolecular interface. In comparison, disordered and lyophilized peptide samples are randomly packed with both parallel and antiparallel alignments. The PG-1 aggregates show significant immobilization of the Phe ring near the beta-turn, further supporting the structural ordering. The intermolecular packing of PG-1 found in the solid state is consistent with its oligomerization in lipid bilayers. This solid-state aggregation approach may be useful for determining the quaternary structure of peptides in general and for gaining insights into the oligomerization of antimicrobial peptides in lipid bilayers in particular.     

Origin of the yellow color of complex nickel oxides

Single-crystal optical absorption spectra of NiO, NiTiO₃, NiWO₄, NiV₂O₆, NiNb₂O₆, Ni₂SiO₄, Ni₃V₂O₈, LiNiPO₄, Li₂Ni₂Mo₃O₁₂, SrNiTeO₆, LiScSiO₄:Ni, MgSiO₃:Ni, and (Mg,Ni)₂SiO₄ are presented for the purpose of comparing the spectra of yellow and green Ni²⁺ compounds. Powder spectra of NiTiO₃, NiWO₄, NiV₂O₆, NiNb₂O₆, and Ni₃V₂O₈ in the ultraviolet region help elucidate the more intense charge transfer bands. Bright yellow color results when Ni²⁺ is in a six-coordinated site significantly distorted from octahedral symmetry. Increased absorption intensity occurs when the metal ion d-d bands are in proximity to an ultraviolet charge transfer band.     

Trehalose-protected lipid membranes for determining membrane protein structure and insertion

Trehalose preserves lipid bilayers during dehydration and rehydration by replacing water to form hydrogen bonds between its own OH groups and lipid headgroups. We compare the lipid conformation and dynamics between trehalose-protected lyophilized membranes and hydrated membranes, to assess the suitability of the trehalose-containing membrane as a matrix for membrane protein structure determination. (31)P spectra indicate that the lipid headgroup of trehalose-protected dry POPC membrane (TRE-POPC) have an effective phase transition temperature that is approximately 50K higher than that of the hydrated POPC membrane. In contrast, the acyl chains have similar transition temperatures in the two membranes. Intramolecular lipid (13)C'-(31)P distances are the same in TRE-POPC and crystalline POPC, indicating that the lipid headgroup and glycerol backbone conformation is unaffected by trehalose incorporation. Intermolecular (13)C-(31)P distances between a membrane peptide and the lipid headgroups are 10% longer in the hydrated membrane at 226 K than in the trehalose-protected dry membrane at 253 K. This is attributed to residual motions in the hydrated membrane, manifested by the reduced (31)P chemical shift anisotropy, even at the low temperature of 226 K. Thus, trehalose lyoprotection facilitates the study of membrane protein structure by allowing experiments to be conducted at higher temperatures than possible with the hydrated membranes.     

Scope of the directed dihydroxylation: application to cyclic homoallylic alcohols and trihaloacetamides

The synthesis and directed dihydroxylation of a range of cyclic alkenes was investigated. Both homoallylic alcohols and homoallylic trihaloacetamides were found to be efficient directing groups, giving rise to good to excellent levels of remote asymmetric induction with OsO4-TMEDA. Interestingly, in all cases examined, trifluoroacetamides were found to be superior to trichloroacetamides as directing groups and an argument is presented which rationalises this observation.     

Highly efficient on-DNA amide couplings promoted by micelle forming surfactants for the synthesis of DNA encoded libraries

DNA encoded libraries (DELs) represent powerful new technology for finding small molecule ligands for proteins and are increasingly being applied to hit finding in medicinal chemistry. Crucial to the synthesis of high quality DELs is the identification of chemical reactions for their assembly that proceed with very high conversion across a range of different substrates, under conditions compatible with DNA-tagged substrates. Many current chemistries used in DEL synthesis do not meet this requirement, resulting in libraries of low fidelity. Amide couplings are the most commonly used reaction in synthesis of screening libraries and also in DELs. The ability to carry out highly efficient, widely applicable amide couplings in DEL synthesis would therefore be highly desirable. We report a method for amide coupling using micelle forming surfactants, promoted by a modified linker, that is broadly applicable across a wide range of substrates. Most significantly, this works exceptionally well for coupling of DNA-conjugated carboxylic acids (N-to-C) with amines in solution, a procedure that is currently very inefficient. The optimisation of separate procedures for coupling of DNA-conjugated acids and amines by reagent screening and statistically driven optimisation is described. The generality of the method is illustrated by the application to a wide range of examples with unprecedented levels of conversion. The utility of the (N-to-C) coupling of DNA-conjugated acids in DEL synthesis is illustrated by the three cycle synthesis of a fully DNA-encoded compound by two cycles of coupling of an aminoester, with intermediate ester hydrolysis, followed by capping with an amine. This methodology will be of great utility in the synthesis of high fidelity DELs.

Highly efficient forward and reverse on-DNA amide couplings were developed exploiting hydrophobic linkers in combination with the micelle forming surfactant TPGS-750M. The method is highly effective for a wide range of substrates in the synthesis of DNA-encoded libraries.     

Cumulant functions in optical coherence theory

Cumulant functions are introduced to describe the statistical state of a radiation field. These functions are simply related to the optical coherence functions but have some interesting features. It is shown that if the cumulant functions of all orders greater than some numberN ₀ vanish then they also vanish for all orders greater than 2. Thermal field is the only field having this property. This property holds whether the field is described by a classical stochastic process or by a quantum density operator. Further the particular operator ordering used in defining these cumulant functions for the quantized field affects only the second order cumulant function. To describe the statistical state of a vector field such as partially polarized or unpolarized radiation, one would need to introduce cumulant tensors.     

Interaction of the antimicrobial peptide dicynthaurin with membrane phospholipids at the air-liquid interface

This paper reports the first study on the interaction of the antimicrobial peptide dicynthaurin with 1,2-dipalmitoyl-glycerophosphatidyl-glycerol investigated in monolayers at the air-liquid interface. The influence of the peptide on the two-dimensional phase behavior of the negatively charged lipid was elucidated by means of pressure-area isotherm measurements, fluorescence microscopy, and grazing incidence X-ray diffraction measurements. The pure peptide forms a stable monolayer at the air-liquid interface up to 30 mN/m as shown for both the monomeric and the dimeric cynthaurins. The peptide lipid interaction was monitored in isotherm measurements showing a strong adsorption of the peptide and stabilization at the interface promoted by the lipid monolayer. The X-ray diffraction measurements in agreement with fluorescence microscopy studies showed that the peptide destabilizes the condensed chain lattice, leading to a complete fluidization of the condensed lipid phase on physiological buffer. The adsorption of the peptide to the negatively charged lipid monolayer and the fluidization of the condensed chain lattice suggest a direct link to the peptides' ability to expand the bacterial membrane that would be relevant for the in vivo mode of action.