High-throughput purification of combinatorial libraries I: a high-throughput purification system using an accelerated retention window approach

We have developed a high-throughput purification system to purify combinatorial libraries at a 50-100-mg scale with a throughput of 250 samples/instrument/day. We applied an accelerated retention window method to shorten the purification time and targeted one fraction per injection to simplify data tracking, lower QC workload, and simplify the postpurification processing. First, we determined the accurate retention time and peak height for all compounds using an eight-channel parallel LC/UV/MS system, and calculated the specific preparative HPLC conditions for individual compounds. The preparative HPLC conditions include the compound-specific gradient segment for individual compounds with a fixed gradient slope and the compound-specific UV or ELSD threshold for triggering a fraction collection device. A unique solvent composition or solvent strength was programmed for each compound in the preparative HPLC in order to elute all compounds at the same target time. Considering the possible deviation of the predicted retention time, a 1-min window around the target time was set to collect peaks above a threshold based on UV or ELSD detection. Dual column preparative instruments were used to maximize throughput. We have purified more than 500 000 druglike compounds using this system in the past 3 years. We report various components of this high-throughput purification system and some of our purification results.     

Studium und Anwendung eines neuen Komplexes von Kupfer mit Salicylsäure

Microchimica Acta - Eine empfindliche spektrophotometrische Methode zur Bestimmung von Kupfer mit Salicyls~iure wurde beschrieben (Molarextinktion ε = 2,4 · 9 105). Die nachweisbaren...     

Microarray-based in vitro evaluation of DNA oligomer libraries designed in silico.

We report on the microarray-based in vitro evaluation of two libraries of DNA oligonucleotide sequences, designed in silico for applications in supramolecular self-assembly, such as DNA computing and DNA-based nanosciences. In this first study which is devoted to the comparison of sequence motif properties theoretically predicted with their performance in real-life, the DNA-directed immobilization (DDI) of proteins was used as an example of DNA-based self-assembly. Since DDI technologies, DNA computing, and DNA nanoconstruction essentially depend on similar prereguisites, in particular, large and uniform hybridization efficiencies combined with low nonspecific cross-reactivity between individual sequences, we anticipate that the microarray approach demonstrated here will enable rapid evaluation of other DNA sequence libraries.     

Modelling and mutation studies on the histamine H1-receptor agonist binding site reveal different binding modes for H1-agonists: Asp116 (TM3) has a constitutive role in receptor stimulation

Summary A modelling study has been carried out, investigating the binding of histamine (Hist), 2-methylhistamine (2-MeHist) and 2-phenylhistamine (2-PhHist) at two postulated agonistic binding sites on transmembrane domain 5 (TM5) of the histamine H₁-receptor. For this purpose a conformational analysis study was performed on three particular residues of TM5, i.e., Lys²⁰⁰, Thr²⁰³ and Asn²⁰⁷, for which a functional role in binding has been proposed. The most favourable results were obtained for the interaction between Hist and the Lys²⁰⁰/Asn²⁰⁷ pair. Therefore, Lys²⁰⁰ was subsequently mutated and converted to an alanine, resulting in a 50-fold decrease of H₁-receptor stimulation by histamine. Altogether, the data suggest that the Lys²⁰⁰/Asn²⁰⁷ pair is important for activation of the H₁-receptor by histamine. In contrast, analogues of 2-PhHist seem to belong to a distinct subclass of histamine agonists and an alternative mode of binding is proposed in which the 2-phenyl ring binds to the same receptor location as one of the aromatic rings of classical histamine H₁-antagonists. Subsequently, the binding modes of the agonists Hist, 2-MeHist and 2-PhHist and the H₁-antagonist cyproheptadine were evaluated in three different seven--helical models of the H₁-receptor built in homology with bacteriorhodopsin, but using three different alignments. Our findings suggest that the position of the carboxylate group of Asp¹¹⁶ (TM3) within the receptor pocket depends on whether an agonist or an antagonist binds to the protein; a conformational change of this aspartate residue upon agonist binding is expected to play an essential role in receptor stimulation.Abbreviations 2-MeHist 2-methylhistamine - 2-PEA 2-pyridyl-ethylamine - 2-PhHist 2-phenylhistamine - CHO Chinese hamster ovary - E_int interaction energy - E_str strain energy - GES global energy structure - gpH₁R guinea pig H₁-receptor - GPCR G-protein coupled receptor - Hist histamine - N proximal nitrogen - N tele nitrogen - TM transmembrane domain - WT wild type     

Inter-method comparison for the determination of antimony and arsenic in peat samples

Four analytical approaches, based on different physical principles, for the determination of antimony (Sb) and arsenic (As) in ancient peat samples were critically evaluated: (a) open vessel digestion/hydride generation-atomic absorption spectrometry (HG-AAS), (b) closed-pressurized digestion in a microwave oven followed by sector field-inductively coupled plasma-mass spectrometry (SF-ICP-MS), (c) digestion in a microwave autoclave and subsequent quadrupole-inductively coupled plasma-mass spectrometry (Q-ICP-MS) measurements and (d) instrumental neutron activation analysis (INAA). The quality control scheme applied, always included the use of adequate plant reference materials to ensure the accuracy and precision of the analytical procedures. Additionally, two internal peat reference materials were analyzed using all four analytical approaches, generally showing good agreement for both elements. Method detection limits for As and Sb provided by all procedures were approximately 5 and 2 ng g⁻¹ which is sufficiently low for the reliable quantification of both elements in ancient, pre-anthropogenic peat samples. A comparison of As and Sb concentrations in a set of peat samples determined by INAA, HG-AAS and SF-ICP-MS revealed that INAA underestimated the values in a systematic manner, whereas HG-AAS and SF-ICP-MS data agreed very well. Best precision of the results was obtained by analytical procedures involving HG-AAS or Q-ICP-MS and varied from 3.6 to 4.3% and 7.1 to 7.5% for As (at about 0.5 μg g⁻¹) and Sb (at about 0.1 μg g⁻¹), respectively. The highest sample throughput (40 samples per run accomplished in 2 h) combined with low risk of sample contamination could be realized in the high-pressure microwave autoclave. The amount of sample required by all approaches was 200 mg, except for INAA which needed at least 25 times more sample mass to achieve comparable detection limits. For the quantification of As and Sb, inductively coupled plasma-mass spectrometry (ICP-MS) was preferred over INAA and HG-AAS, mainly because (a) less sample is needed and (b) As and Sb can be determined simultaneously. In addition, ICP-MS offers the possibility to measure concurrently a wide range of other elements which also are of environmental interest.     

Hydrogen analysis in solid samples by utilizing He metastable atoms induced by TEA CO2 laser plasma in He gas at 1 atm

A TEA CO₂ laser (350 mJ–1.5 J, 10.6 μm, 200 ns, 10 Hz) was focused onto a metal sub-target under He as host gas at 1 atmospheric pressure with a small amount of impurity gas, such as water and ethanol vapors. It was found that the TEA CO₂ laser with the help of the metal sub-target is favorable for generating a strong, large volume helium gas breakdown plasma at 1 atmospheric pressure, in which the helium metastable-excited state was then produced overwhelmingly. While the metal sub-target itself was never ablated. The helium metastable-excited state produced after the strong helium gas breakdown plasma was considered to play an important role in exciting the atoms. This was confirmed by the specific characteristics of the detected H emission, namely the strong intensity with low background, narrow spectral width, and the long lifetime. This technique can be used for gas and solid samples analysis. For nonmetal solid analysis, a metal mesh was introduced in front of the nonmetal sample surface to help initiation of the helium gas breakdown plasma. For metal sample, analysis can be carried out by combining the TEA CO₂ laser and an Nd–YAG laser where the Nd–YAG laser is used to ablate the metal sample. The ablated atoms from the metal sample are then sent into the region of helium gas breakdown plasma induced by the TEA CO₂ laser to be excited through the helium metastable-excited state. This technique can be extended to the analysis of other elements, not limited only to hydrogen, such as halogens.     

A molecular dynamics study of the pentacyclo-undecane cage amino acid tripeptide

In this paper, we report on the conformational profile of the pentacyclo-undecane (PCU) cage tripeptide carried out by molecular dynamics (MD) simulation using water as an explicit solvent. The MD solution phase studies carried on the model peptide analogues (A)=Ac–Ala–Ala–Ala–NHMe; (B)=Ac–Cage–Cage–Cage–NHMe; (C)=Ac–Ala–Cage–Ala–NHMe and (D)=Ac–Ala–Pro–Ala–NHMe, are used as a complimentary technique to the corresponding gas phase simulated annealing (SA) study previously carried out in our laboratory. No significant structural changes were observed over the MD trajectories. However, the results reported here provide further evidence that the (PCU) cage amino acid exhibits C_7eq, C_7aq, _R and _L conformations, and the theoretical results suggest that the PCU cage amino acid is a strong β-turn inducer. These results support the prediction that when the PCU cage residues are in the (i) and (i+2) positions, the β-turn can be extended in either direction to form anti-parallel β-pleated sheets, thereby forming the basis of the mechanism for the folding back of the chain in a cross-β-turn structure.     

Practical asymmetric enzymatic reduction through discovery of a dehydrogenase-compatible biphasic reaction media

[reaction: see text] An enzyme-compatible biphasic reaction media for the asymmetric biocatalytic reduction of ketones with in situ cofactor regeneration has been developed. In this biphasic reaction media, which is advantageous for reactions at higher substrate concentrations, both enzymes (alcohol dehydrogenase and FDH from Candida boidinii) remain stable. The reductions with poorly water-soluble ketones were carried out at substrate concentrations of 10-200 mM, and the optically active (S)-alcohols were formed with moderate to good conversions and with up to >99% ee.     

Coligand role in the NHC nickel catalyzed C–F bond activation: investigations on the insertion of bis(NHC) nickel into the C–F bond of hexafluorobenzene

The reaction of [Ni(Mes₂Im)₂] (1) (Mes₂Im = 1,3-dimesityl-imidazolin-2-ylidene) with polyfluorinated arenes as well as mechanistic investigations concerning the insertion of 1 and [Ni(ⁱPr₂Im)₂] (1ipr) (ⁱPr₂Im = 1,3-diisopropyl-imidazolin-2-ylidene) into the C–F bond of C₆F₆ is reported. The reaction of 1 with different fluoroaromatics leads to formation of the nickel fluoroaryl fluoride complexes trans-[Ni(Mes₂Im)₂(F)(Ar^F)] (Ar^F = 4-CF₃-C₆F₄2, C₆F₅3, 2,3,5,6-C₆F₄N 4, 2,3,5,6-C₆F₄H 5, 2,3,5-C₆F₃H₂6, 3,5-C₆F₂H₃7) in fair to good yields with the exception of the formation of the pentafluorophenyl complex 3 (less than 20%). Radical species and other diamagnetic side products were detected for the reaction of 1 with C₆F₆, in line with a radical pathway for the C–F bond activation step using 1. The difluoride complex trans-[Ni(Mes₂Im)₂(F)₂] (9), the bis(aryl) complex trans-[Ni(Mes₂Im)₂(C₆F₅)₂] (15), the structurally characterized nickel(i) complex trans-[Ni^I(Mes₂Im)₂(C₆F₅)] (11) and the metal radical trans-[Ni^I(Mes₂Im)₂(F)] (12) were identified. Complex 11, and related [Ni^I(Mes₂Im)₂(2,3,5,6-C₆F₄H)] (13) and [Ni^I(Mes₂Im)₂(2,3,5-C₆F₃H₂)] (14), were synthesized independently by reaction of trans-[Ni(Mes₂Im)₂(F)(Ar^F)] with PhSiH₃. Simple electron transfer from 1 to C₆F₆ was excluded, as the redox potentials of the reaction partners do not match and [Ni(Mes₂Im)₂]⁺, which was prepared independently, was not detected. DFT calculations were performed on the insertion of [Ni(ⁱPr₂Im)₂] (1ipr) and [Ni(Mes₂Im)₂] (1) into the C–F bond of C₆F₆. For 1ipr, concerted and NHC-assisted pathways were identified as having the lowest kinetic barriers, whereas for 1, a radical mechanism with fluoride abstraction and an NHC-assisted pathway are both associated with almost the same kinetic barrier.

A combined experimental and theoretical study on the mechanism of the C–F bond activation of C₆F₆ with [Ni(NHC)₂] is provided.     

Simulation of 59Co NMR chemical shifts in aqueous solution

59Co chemical shifts were computed at the GIAO-B3LYP level for [Co(CN)6]3-, [Co(H2O)6]3+, [Co(NH3)6]3+, and [Co(CO)4]- in water. The aqueous solutions were modeled by Car-Parrinello molecular dynamics (CPMD) simulations, or by propagation on a hybrid quantum-mechanical/molecular-mechanical Born-Oppenheimer surface (QM/MM-BOMD). Mean absolute deviations from experiment obtained with these methods are on the order of 400 and 600 ppm, respectively, over a total delta(59Co) range of about 18,000 ppm. The effect of the solvent on delta(59Co) is mostly indirect, resulting primarily from substantial metal-ligand bond contractions on going from the gas phase to the bulk. The simulated solvent effects on geometries and delta(59Co) values are well reproduced by using a polarizable continuum model (PCM), based on optimization and perturbational evaluation of quantum-mechanical zero-point corrections.