To realize the full potential of combinatorial chemistry-based drug discovery, generic and efficient tools must be developed that apply the strengths of diversity-oriented chemical synthesis to the identification and optimization of lead compounds for disease-associated protein targets. We report an affinity selection-mass spectrometry (AS-MS) method for protein-ligand affinity ranking and the classification of ligands by binding site. The method incorporates the following steps: (1) an affinity selection stage, where protein-binding compounds are selected from pools of ligands in the presence of varying concentrations of a competitor ligand, (2) a first chromatography stage to separate unbound ligands from protein-ligand complexes, and (3) a second chromatography stage to dissociate the ligands from the complexes for identification and quantification by MS. The ability of the competitor ligand to displace a target-bound library member, as measured by MS, reveals the binding site classification and affinity ranking of the mixture components. The technique requires no radiolabel incorporation or direct biochemical assay, no modification or immobilization of the compounds or target protein, and all reaction components, including any buffers or cofactors required for protein stability, are free in solution. We demonstrate the method for several compounds of wide structural variety against representatives of the most important protein classes in contemporary drug discovery, including novel ATP-competitive and allosteric inhibitors of the Akt-1 (PKB) and Zap-70 kinases, and previously undisclosed antagonists of the M(2) muscarinic acetylcholine receptor, a G-protein coupled receptor (GPCR). The theoretical basis of the technique is analyzed mathematically, allowing quantitative estimation of binding affinities and, in the case of allosteric interaction, absolute determination of binding cooperativity. The method is readily applicable to high-throughput screening hit triage, combinatorial library-based affinity optimization, and developing structure-activity relationships among multiple ligands to a given receptor.

Graphene quantum dots (GQDs) have been prepared for the first time using raw plant leaf extracts of Neem (Azadirachta indica) and Fenugreek (Trigonella foenum-graecum) by a facile, hydrothermal method at 300 1C for 8 hours in water, without the need of any passivizing, reducing agents or organic solvents. High resolution transmission electron microscope studies showed that the average sizes of the GQDs from Neem (N-GQDs) and Fenugreek (F-GQDs) were 5 and 7 nm respectively. N-GQDs and F-GQDs exhibit high quantum yields of 41.2% and 38.9% respectively. Moreover, the GQDs were utilized to prepare a white light converting cap based on the red-green-blue (RGB) color mixing method.

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Quantized magnetotransport is observed in 5.6 × 5.6 mm2 epitaxial graphene devices, grown using highly constrained sublimation on the Si-face of SiC(0001) at high temperature (1900 °C). The precise quantized Hall resistance of Rxy=h2e2 is maintained up to record level of critical current Ixx = 0.72 mA at T = 3.1 K and 9 T in a device where Raman microscopy reveals low and homogeneous strain. Adsorption-induced molecular doping in a second device reduced the carrier concentration close to the Dirac point (n ≈ 1010 cm−2), where mobility of 18760 cm2/V is measured over an area of 10 mm2. Atomic force, confocal optical, and Raman microscopies are used to characterize the large-scale devices, and reveal improved SiC terrace topography and the structure of the graphene layer. Our results show that the structural uniformity of epitaxial graphene produced by face-to-graphite processing contributes to millimeter-scale transport homogeneity, and will prove useful for scientific and commercial applications.

We report the dispersed fluorescence spectra of the linear and the previously well-studied T-shaped isomers of Ar-I 2 following B←X optical excitation for v pump ϭ16-26, below the I 2 dissociation limit. The linear isomer has a continuum excitation spectrum. For excitation at the highest pumping energy (v pump ϭ26), the product vibrational state distribution is nearly identical to that observed for excitation above the I 2 (B) dissociation limit; it shows a broad, nearly Gaussian distribution of I 2 (B) vibrational states, with about 22% of the available excess energy deposited in translation of the ArϩI 2. This gives direct evidence that the ''one-atom cage'' effect seen above the I 2 (B) dissociation limit is attributable to the linear Ar-I 2 isomer. The product vibrational state distribution becomes increasingly Poisson for decreasing excitation energies, and only about 7% of the excess energy is deposited in translation for v pump ϭ16. The bond energy in the linear isomer is determined from the spectra, 170(Ϯ1.5)рD 0 Љ(linear Ar-I 2 (X))р174(Ϯ1.5) cm Ϫ1. A bond energy of D 0 Љ(T-shaped Ar-I 2 (X))ϭ142Ϯ15 cm Ϫ1 is estimated based on the linear to T-shaped population ratio observed in the beam, which is about 90 cm Ϫ1 smaller than that determined from fluorescence spectra. We suggest that electronic quenching in the T-shaped isomer is nearly 100% for the highest vibrational level produced by vibrational predissociation.

An affinity-based mass spectrometry screening technology was used to identify novel binders to both nonphosphorylated and phosphorylated ERK2. Screening of inactive ERK2 identified a pyrrolidine analogue 1 that bound to both nonphosphorylated and phosphorylated ERK2 and inhibited ERK2 kinase activity. Chemical optimization identified compound 4 as a novel, potent, and highly selective ERK1,2 inhibitor which not only demonstrated inhibition of phosphorylation of ERK substrate p90RSK but also demonstrated inhibition of ERK1,2 phosphorylation on the activation loop. X-ray cocrystallography revealed that upon binding of compound 4 to ERK2, Tyr34 undergoes a rotation (flip) along with a shift in the poly-Gly rich loop to create a new binding pocket into which 4 can bind. This new binding mode represents a novel mechanism by which high affinity ATP-competitive compounds may achieve excellent kinase selectivity.

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