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Lipid A-diphosphate is associated with lethal endotoxicity, specific immune responses, and plays a key role during infection with microorganisms or viruses. Periodic self-assembled lipid A-diphosphate structures were investigated by light scattering, small-angle X-ray scattering (SAXS) in aqueous dispersions, transmission electron microscopy (TEM), and small-area electron diffraction. SAXS experiments are consistent with equilibrium phase structures that appear as crystal variants of dodecahedra and icosahedra depending on lipid A-phosphate composition. The lipid A-diphosphate form factors were also calculated by nonlinear parameters using an efficient Bayesian parameter estimation. The phases for the lipid A-diphosphate and their mixture components show 12-fold or sixfold diffraction symmetry. The two-dimensional (2D) cross-sectional diffraction patterns are consistent with (6.6.6), (4.6.12), and (3.3.4.3.4) Archimedean tiling. Changes of the ratio of the lipid A-diphosphate variants in the quasicrystal are accompanied with a change of the ratio of the equilateral triangles and squares in the unit cells. The predominant 2D tiling structures are the honeycomb-type structure with a tiling pattern, where each hexagonal domain contacts the other six domains.

Small-angle x-ray scattering and quasi-elastic light scattering measurements were conducted to analyze correlations between the structure and short-time dynamics of tyrocidine B·hydrochloride (TrcB) in ethanol, acetonitrile, and (R,S)-2-methylbutanol dispersions in the presence of 20.0% (v/v) water at 278 K and 298 K. The three TrcB dispersions exhibited peak position shifts that varied with the volume fraction. The experimental data were fitted to a model that considered the effective interaction potential, short-range attraction, and long-range repulsion. This model of repulsively interacting single TrcB particles is incompatible with the presence of equilibrium aggregate phases. The self-diffusion coefficient at the short-time limit (Dsh) decreased more as the TrcB concentration increased than one would expect for a corresponding hard-sphere or charged particle at the same volume fraction. At low volume fractions, the system consisted of monomers, dimers, and trimers. At high TrcB volume fractions, the main particles were larger aggregates. The collective diffusion coefficient, Dc, was constant when Q > Qc, where Qc is the position of the interference peak, which implies that there were no inter-monomer TrcB oligomer dynamics. This is because Dsh/D0 decayed much more quickly than the TrcB monomer as a function of the volume fraction. In vitro experiments revealed that antimicrobial activities were preserved at all volume fractions notwithstanding the presence of various oligomers.

The presence of lipopolysaccharides (LPSs) in Gram-negative bacteria, particularly the lipophilic part of LPSs, lipid A-diphosphate, is associated with lethal endotoxicity, pyrogenicity, specific immune response, triggering of cascade of cellular mediators, septic shock, and other life-threatening actions in life. A detailed knowledge of the spatial structure in solution and the packing of nontoxic lipid A phosphates isolated from humans in the self-assembly will aid in the physical understanding of these interactions. The high incidence and increased severity have resulted in growing interest to develop new vaccines on the basis of lipid A-phosphate approximants. Using the distribution of various lipid A-phosphate elements, tiles may be designed with different patterns of toxic and nontoxic lipid A-phosphate elements on each edge of the pentagon, and each with a distinct complementary edge mate, similar to the design of programmed assembly of coded tiles. Assuming that the interface formed by any complementary pair is isostructural with all other potential structures, then any combination of 12 tiles with appropriately matched edges may form a stable dodecahedra structure. This possibility of building a hierarchical self-assembly of lipid A-phosphates with various constituents as analogues is capable of producing an enormous diversity of patterned assemblies. Notably, the importance of electrostatic stabilized nanocrystals at predefined pH and ionic strength enables the lipid A-phosphate system to avoid random aggregate cluster formation.

Surface-tension-induced liquid-crystal growth of monomeric lipid A-diphosphate in aqueous dispersions is reported as a function of concentration, (c), and temperature, (T), and at low ionic strength (10(-3) M). As the temperature was varied, a solid-liquid transition was revealed in the surface layer at a fixed lipid A-diphosphate bulk concentration. Here, the development of different two-dimensional (2-d) faceted crystal morphologies was observed and, as growth proceeded, these faceted 2-d crystals became unstable. Selected area electron microscopy diffraction (SAED) and X-ray diffraction (XRD) measurements of the faceted 2-d crystalline lipid A-diphosphate layers exhibited a pseudohexagonal molecular arrangement. The crystalline layer was a smectic F, S(F), phase below the critical temperature, T(C), and a smectic I, S(I), phase above T(C) (15 °C). Both phases could be described in terms of the same C-centered monoclinic unit cell. The in-plane order extended for a limited distance although the layers were coupled. The analysis of the SAED patterns revealed short-range order in the S(F) phase (5-15 °C), but long-range order in the S(I) phase, for the temperature range 15-30 °C. The observed 2-d solid hexatic phase and the 2-d liquid hexatic phase had correlation lengths of 220 Å. This, the hexatic phase, displayed short-range in-plane positional order and quasi long-range, sixfold bond-orientational order. The S(I) phase showed long-range order characteristics of a hexatic 2-d crystal. The two-, four-, or six-layer crystalline lipid A-diphosphate films exhibited 2-d hexatic order and 6n-fold bond-orientational order. These films did not evolve into the S(F) phase, demonstrating that the two phases were thermodynamically distinct. A finite tilt angle of φ = 15° was calculated for the lipid A-diphosphate molecule; the tilt was toward the small side of the rectangular 2-d lattice. The constraint of six close-packed acyl chains in two distinct phases with the same symmetry suggests that the S(F) → S(I) transition was first-order.

The nature of the fluid phase transitions of charged-stabilized spherical lipid A-diphosphate clusters in aqueous dispersions was explored using a combination of small-angle X-ray scattering (SAXS) and electron microscopy. In contrast to previous studies, rather than removing NaCl, NaOH was added to the dispersions to promote crystallization. The fluid phase experiments were carried out employing titrations with mM·L(-1) NaOH (c(S)), along with variations in the particle-number density, n. When c(S) was increased, a new fluid disordered phase of self-assembled lipid A-diphosphate was encountered, followed by a crystalline bcc phase and then a new fluid phase containing 70 nm lipid A-diphosphate particles. The bcc crystal structure found in this regime had a lattice constant of 35.6 nm. By varying c(S) (mM L(-1)), it was possible to determine the effective charge, z(eff), for various n values and the screening parameter, k, for the excess electrolyte. For sufficiently large values of n, lipid A-diphosphate crystallized because of an increase in z(eff) at a constant c(S). When the c(S) was increased, the crystals melted with little change in z(eff). The existence of a bcc-fluid phase transition for different values of c(S) was supported by applying the Debye correlation function to the obtained data. An increase in c(S) enhanced interparticle interaction and attraction. The effective charge and k accounted for counterion condensation and many-body effects. If the effective charge determined from scattering measurements was used in the simulations, the equilibrium phase boundaries were consistent with predicted universal melting-line simulations. At any particle-number densities, n, when the melting line was reached, 70 nm clusters were formed.

The well-defined structure of lipid A-diphosphate in aqueous solutions provides a way of observing the formation of calcium carbonate crystals. The crystals are either tetrahedral or rhombohedral calcite at a volume fraction of phi = 5.4 x 10 (-4) at pH 5.8 or the vaterite polymorph of CaCO(3) at a volume fraction of phi = 7.8 x 10 (-4) at pH 5.8. In both cases, nucleation, adsorption pH, and the shape-dependent template of lipid A-diphosphate control the formation of the calcite and vaterite.

Small-angle X-ray-scattering, light-scattering, and electron microscope experiments were used to determine the phase transitions of colloidal lipid A-diphosphate aqueous dispersions. The phases detected were a correlated liquid phase, a face-centered cubic (Fd3m) and a body-centered cubic (Im3m) colloidal crystal phase and a new glass phase. These experimentally determined phases were shown to be in accord with theoretically predicted equilibrium phases.

A comprehensive structural characterization of the colorless and yellow forms of N-hydroxyphthalimide (NHP), the deuterated form (NDP), and the ethoxylated form (ethoxy-NHP) has been carried out using single-crystal X-ray diffraction, FTIR and Raman spectroscopies, and scanning electron microscopy. Both NHP and NDP forms crystallize in the monoclinic space group (P21/c, No. 14). The various forms of NHP differ in the way in which the molecules adjoin one another through their N-hydroxyl groups and how the carbonyls of the isoindole-1,3-dione ring differ through intermolecular hydrogen bonding. Although the hydrogen bonding about the b axis is virtually the same, the isoindole-1,3-dione ring experiences different twists for the two NHP forms. Both the colorless and yellow forms of NHP exhibit strong intermolecular hydrogen bonding between O(3) and H(1). In the yellow form, the N-hydroxyl group is significantly out of the plane (approximately 1.19 degrees ), but the N-hydroxyl group in the colorless form is only approximately 0.06 degrees out of the plane. Both forms of NHP reveal an infinite chain of intermolecular hydrogen-bonded molecules in the direction of the b axis; however, the molecules are ordered differently within the unit cells. The hydrogen-bond geometry for the yellow form of NHP is O(2)-H(1)...O(3), with an angle of 185 degrees , intermolecular distances of O(2)...O(3) = 2.68 A and H(1)...O(3) = 1.70 A, and an intramolecular hydrogen bond of O(1)...H(1) = 1.17 A. The colorless form of NHP shows an intermolecular hydrogen-bond geometry between O(3) and H(1) with a distance of 1.78 A; the O(2)-O(3) distance is 2.71 A. The O(2)-H(1)...O(3) angle is 159 degrees, and the intramolecular distance is O(1)...H(1) = 0.97 A. The N-ethoxy derivative of NHP crystallizes in an orthorhombic space group (Pnma, No. 62) and exhibits no hydrogen bonding, displaying a strong head-to-tail stacking of the planar rings along the needle axis direction.

The materials studied in this investigation were aqueous solutions (0.02-25.0 mM) of the salts of alkali metal ion (Me(+)) and 2-nitrophenol (2-NP). In the investigation, small-angle X-ray scattering, wide-angle X-ray scattering, and membrane-pressure osmometry were used to study the 2-NP-Me(+) molecular salt structures and the onset of crystallization as a function of concentration and temperature. The experimental methods used to examine the 2-NP-Me(+) molecular salt complexes provided corroborative evidence for the existence of spherical clusters with hydrodynamic diameters between ∼12 Å (Li) and 14 Å (Cs). Guinier plots of the zero-angle scattering peak were characteristic of the scattering from lamellae-like shapes with thicknesses of ∼290 Å. Tetramer and pentamer 2-NP-Me(+) molecular clusters for Me(+) = Li, Na, K, and Rb were assembled from four or five 2-NP molecules bound to a central alkali metal ion. The coordination symmetry around the six coordinated Li(+), Na(+), and K(+) ions was that of a trigonal prism (D3h), with an octahedral arrangement (D2h). The Rb(+) also revealed six-coordinate geometry and the central Rb(+) ion adopted an octahedral arrangement (D2h). The eight-coordinated Cs(+) ions with six 2-NP ligands were characteristic of a square antiprism (D4d). The square antiprism was the outcome of leaving two o-nitro groups and two phenolic oxygens being left intermolecularly uncoordinated to the Cs(+) ion. The 2-NP residues were strictly planar and contained short non-bonded intramolecular distances. van der Waals forces were present between the adjacently stacked phenyl rings. No water molecules were involved as ligands for any of the 2-nitrophenol-Me(+) complexes.

Dilute electrostatically stabilized aqueous solutions of hexa-acylated (C14) lipid A diphosphate from Escherichia coli form stable and regularly shaped colloidal crystals in a size range of approximately 50-1000 nm in width and 50-100 nm in thickness. The formation of these nanocrystals occurs over a range of volume fractions between 3.5 × 10-3 and 1.2 × 10-2 and at a low ionic strength, ~10-5. The shape of these crystals appears to be cubic or rhombohedral, and when exposed to the electron beam, these fragile nanocrystals are easily damaged. Electron diffraction patterns obtained from single particles reveal that they are orientated (001) crystals that conform to a trigonal or hexagonal unit cell (a = 3.65 ± 0.07 nm and c = 1.97 ± 0.04 nm), revealing crystal-like pore walls that exhibit structural periodicity with a spacing of 0.65 nm and are at least four times the size of the unit cell adopted by lipid A diphosphate.

It has been possible to prepare electrostatically stabilized aqueous dispersions of lipid A (diphosphate) particles of low polydispersity at low ionic strength (1-10 mM NaCl) over a range of volume fractions of 1.5 × 10-4 < < 5.75 × 10-4 (25 C). These suspensions have been characterized by transmission electron microscopy, light scattering, osmotic pressure measurements, and small-angle X-ray scattering experiments at 25 C. All four measurements yielded independent values for particle sizes, weighted-average molecular weights, number-average molecular weights, and particle surface charge. The mean values obtained are = 37.59 ± 0.75 nm, = 24.89 ± 0.88 nm, = (10.55 ± 0.78) × 106 g/mol, = (9.81 ± 0.90) × 106 g/mol, and the effective surface charge Z* = (756 ± 85). Very good experimental agreement is found for the directly measured osmotic pressure values and those determined from light scattering and small-angle X-ray scattering measurements as a function of volume fraction, , by applying liquid-state theory models. Using the particle parameters for the lipid A (diphosphate) system as determined, the scattering functions and the osmotic pressures can be compared as a function of volume fraction with no adjustable parameters. The ordering of lipid A in solution revealed a body-centered cubic (bcc) type lattice (a = 36.14 nm) at volume fractions of 3.75 × 10-4 < < 4.15 × 10-4, whereas at volume fractions of 4.15 × 10-4 < < 5.75 × 10-4 in the presence of 1.0 mM NaCl a face-centered cubic (fcc) lattice type (a = 57.25 nm) was observed. Small-angle X-ray scattering experiments also indicate the presence of long-ranged order at 1.0 mM or at 10.0 mM NaCl for lipid A dispersions of 3.75 × 10-4 < < 5.75 × 10-4.

Electrostatically stabilized aqueous dispersions of nm-sized free lipid A particles at low volume fractions (1.0×10-4 ρTEM=8.0±0.6%) yield similar values for the particle size and particle size distribution ( ρQELS=10.9±0.75 %), whereas conductivity and acid-base titrations estimate surface chemical parameters (dissociation constant, ionizable sites, and Stern capacitance). Effective particle charges were determined by fits of the integral equation calculations of the polydisperse static structure factor, S(Q), to the light scattering data. Using the particle properties as determined from these experiments, the polydisperse structure factor, S(Q), was calculated as a function of volume fraction, θ, which was found to be consistent with a S(Q) dependence on the number particle density. It can be concluded that, at low volume fractions and low ionic strength, the light scattering data are well represented by a Poisson-Boltzmann model (PBC) of fluid-like ordering of free lipid A in aqueous solution. We find that the light scattering data of this dispersion are best described by a model where only a small fraction of the ionizable phosphate groups is dissociated at neutral pH. Finally, light scattering studies of lipid A dispersions of volume fractions of 3.9×10-4