Activity-based probe for histidine kinase signaling

Bacterial two-component systems (TCSs) are signaling pathways composed of two proteins: a histidine kinase (HK) and a response regulator (RR). Upon stimulation, the HK autophosphorylates at a conserved histidine. The phosphoryl group is subsequently transferred to an aspartate on an RR, eliciting an adaptive response, often up- or downregulation of gene expression. TCS signaling controls many functions in bacteria, including development, virulence, and antibiotic resistance, making the proteins involved in these systems potential therapeutic targets. Efficient methods for the profiling of HKs are currently lacking. For direct readout of HK activity, we sought to design a probe that enables detection of the phosphotransfer event; however, analysis of the phosphohistidine species is made difficult by the instability of the P-N bond. We anticipated that use of a γ-thiophosphorylated ATP analogue, which would yield a thiophosphorylated histidine intermediate, could overcome this challenge. We determined that the fluorophore-conjugated probe, BODIPY-FL-ATPγS, labels active HK proteins and is competitive for the ATP binding site. This activity-based probe provides a new strategy for analysis of TCSs and other HK-mediated processes and will facilitate both functional studies and inhibitor identification.     

Synthesis of a functionalized oxabicyclo[2.2.1]-heptene-based chemical library

The 7-oxabicyclo[2.2.1]heptene ring system is a common structural motif in many pharmacologically interesting molecules. We recognized the potential to employ this highly oxygenated and conformationally-restricted scaffold in diversity-oriented synthesis to generate a library of non-chiral but topologically complex compounds. Herein, we report the synthesis and biological evaluation of two 96-member tricyclic libraries containing the oxabicyclo[2.2.1]heptene framework using acetal formation as the key step.     

Synthesis of an 8-pentafluorosulfanyl analog of the antimalarial agent mefloquine

The 8-SF₅ analog of mefloquine was synthesized in nine steps from commercially available starting materials and in five steps from a novel ortho-SF₅-substituted aniline intermediate.     

Structural studies of Vgamma2Vdelta2 T cell phosphoantigens

Human gammadelta T cells containing the Vgamma2Vdelta2 (Vgamma9Vdelta2) T cell receptor are stimulated by a broad variety of small, phosphorus-containing antigenic molecules called phosphoantigens. The structures of several species present in both Mycobacteria (TUBags1-4) and in Escherichia coli have been reported to contain a formyl-alkyl diphosphate core. Here we report the synthesis of the lead member of the series, 3-formyl-1-butyl diphosphate. This compound has low activity for gammadelta T cell stimulation, unlike its highly active isomer (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate, necessitating a revision of the structure of TUBag1. Likewise, the structure of the species identified as the pentyl analog (TUBag 2) is revised to 6-phosphogluconate. These results indicate that neither TUBag1 nor the m/e 275 species proposed for TUBag2 are 3-formyl-1-alkyl diphosphates, leading to the conclusion that none of the natural phosphoantigens (TUBags1-4) possess the structures reported previously.     

Exchange-hole dipole moment and the dispersion interaction: high-order dispersion coefficients

In recent publications [A. D. Becke and E. R. Johnson, J. Chem. Phys. 122, 154104 (2005); E. R. Johnson and A. D. Becke 123, 024101 (2005)] we have demonstrated that the position-dependent dipole moment of the exchange hole can be used to generate dispersion interactions between closed-shell systems. Remarkably accurate C6 coefficients and intermolecular potential-energy surfaces can be obtained from Hartree-Fock occupied orbitals and polarizability data alone. In the present work, our model is extended to predict C8 and C10 coefficients as well. These higher-order coefficients are obtained as easily as C6 and with comparable accuracy.     

Spectral reciprocity and matrix representations of unbounded operators

We study a family of unbounded Hermitian operators in Hilbert space which generalize the usual graph-theoretic discrete Laplacian. For an infinite discrete set X, we consider operators acting on Hilbert spaces of functions on X, and their representations as infinite matrices; the focus is on ?²(X), and the energy space HE. In particular, we prove that these operators are always essentially self-adjoint on ?²(X), but may fail to be essentially self-adjoint on HE. In the general case, we examine the von Neumann deficiency indices of these operators and explore their relevance in mathematical physics. Finally we study the spectra of the HE operators with the use of a new approximation scheme.     

Baicalin Protects Keratinocytes from Toll‐like Receptor‐4 Mediated DNA Damage and Inflammation Following Ultraviolet Irradiation

UVB radiation contributes to both direct and indirect damage to the skin including the generation of free radicals and reactive oxygen species (ROS), inflammatory responses, immunosuppression and gene mutations, which can ultimately lead to photocarcinogenesis. A plant‐derived flavonoid, baicalin, has been shown to have antioxidant, anti‐inflammatory and free radical scavenging activities. Previous studies from our laboratory have shown that in murine skin, Toll‐like receptor‐4 (TLR4) enhanced both UVB‐induced DNA damage and inflammation. The aim of this study was to investigate the efficacy of baicalin against TLR4‐mediated processes in the murine keratinocyte PAM 212 cell line. Our results demonstrate that treating keratinocytes with baicalin both before and after UV radiation (100 mJ cm^?2) significantly inhibited the level of intracellular ROS and decreased cyclobutane pyrimidine dimers and 8‐Oxo‐2′‐deoxyguanosine (8‐oxo‐dG)—markers of DNA damage. Furthermore, cells treated with baicalin demonstrated an inhibition of TLR4 and its downstream signaling molecules, MyD88, TRIF, TRAF6 and IRAK4. TLR4 pathway inhibition resulted in NF‐κB inactivation and down‐regulation of iNOS and COX‐2 protein expression. Taken together, baicalin treatment effectively protected keratinocytes from UVB‐induced inflammatory damage through TLR pathway modulation.     

Optically Pure,Monodisperse cis‐Oligodiacetylenes: Aggregation‐Induced Chirality Enhancement

Conformational changes in the conjugated backbone of poly‐ and oligodiacetylenes (PDAs and ODAs) play an important role in determining the electronic properties of these compounds. At the same time, conformational changes can also result in a folded structure that shows helical chirality. Using D ‐camphor as a chiral building block, we have designed a high‐yielding, iterative synthesis of monodisperse, optically pure cis‐oligodiacetylenes (ODAs). cis‐ODAs up to the tridecamer have been formed, which is the longest monodisperse cis‐ODA reported to date. UV/Vis spectroscopy suggests a large effective conjugation length in THF, likely the result of a linear, planar conformation in this solvent. High‐resolution STM/AFM measurements of the nonamer cast from THF onto HOPG show a linear structure. In iPrOH, circular dichroism (CD) spectra suggest the formation of chiral aggregates for ODAs with at least nine D ‐camphor units, based on a strong CD response.     

Substrate surface energy dependent morphology and dewetting in an ABC triblock copolymer film

A gradient combinatorial approach was used to examine the effect of substrate surface energy on the morphology and stability of films of a poly(isoprene-b-styrene-b-ethylene oxide) triblock copolymer that exhibits an alternating gyroid morphology in the bulk. Atomic force microscopy data across our surface energy (water contact angle) library suggest a transformation to predominantly surface parallel lamellae with an antisymmetric ordering. For substrate water contact angles below 70 degrees the film exhibited autophobic dewetting from an adsorbed half-period triblock copolymer monolayer at longer annealing times. X-ray photoelectron spectroscopy and near edge X-ray absorption fine structure analysis along gradient specimens indicated that the substrate surface energy governed the composition profile of the monolayer, and this variation in chemical expression was key to whether the film was stable or autophobically dewet. These observations demonstrate that enthalpic interactions, in addition to entropic considerations, can play a major role in autophobic dewetting of block copolymer films.     

Improved density functionals for water

The accuracy of existing density functional methods for describing the noncovalent interaction energies in small water clusters is investigated by testing 25 density functionals against a data set of 28 water dimers and 8 water trimers whose structures are taken from the literature and from simulations. The most accurate functionals are found to be PW6B95 with a mean unsigned error of 0.13 kcal/mol and MPWB1K and B98 with mean unsigned errors of 0.15 kcal/mol; the best functional with no Hartree-Fock exchange is mPWLYP, which is a GGA with a mean unsigned error of 0.28 kcal/mol. In comparison, the most popular GGA functionals, PBE and BLYP, have mean unsigned errors of 0.52 and 1.03 kcal/mol, respectively. Since GGAs are very cost efficient for both condensed-phase simulations and electronic structure calculations on large systems, we optimized four new GGAs for water. The best of these, PBE1W and MPWLYP1W, have mean unsigned errors of 0.12 and 0.17 kcal/mol, respectively. These new functionals are well suited for use in condensed-phase simulations of water and ice.