Automatic mass spectrometry method development for drug discovery: application in metabolic stability assays

High-throughput metabolic screening has been requested routinely to keep pace with high-throughput organic synthesis. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a fast gradient has become the method of choice for the task due to its sensitivity and selectivity. We have developed an automated system that consists of a robotic system for in vitro incubation and a commercially available software package for automatic MS/MS method development. A short, generic LC gradient and MS conditions that are applicable to most compounds have been developed to minimize the method development time and data analysis. This system has been used to support a number of in vitro screening assays in early drug discovery phase including microsomal stability and protein binding.     

Characterization of acridone dyes for use in four-decay detection in DNA sequencing

Four acridone dyes and dye-labeled primers were characterized for use in four-decay DNA sequencing. In the four-decay scheme, fluorescence lifetime replaces spectral ("color") selectivity for distinguishing between four base-specific labels in a single-lane capillary electrophoresis (CE) separation of the DNA fragments. Prior to the introduction of the acridone dyes, a major obstacle to four-decay detection was the lack of four suitable dyes with resolvable lifetimes. The four acridone dyes, whether free in solution or tethered to DNA primer, exhibit significant differences among their lifetimes and are well-suited to use together in four-decay sequencing. The lifetimes of the four dye-labeled DNA primers that were sequentially injected and detected on-the-fly in a 2% POP6 sequencing gel were 4, 6, 11 and 14 ns. A 405 nm violet laser diode provides optimal excitation of the four dyes.     

Use of an automated image processing program to quantify recombinant adenovirus particles.

Electron microscopy has a pivotal role as an analytical tool in pharmaceutical research. However, digital image data have proven to be too large for efficient quantitative analysis. We describe here the development and application of an automated image processing (AIP) program that rapidly quantifies shape measurements of recombinant adenovirus (rAd) obtained from digitized field emission scanning electron microscope (FESEM) images. The program was written using the macro-recording features within Image-Pro Plus software. The macro program, which is linked to a Microsoft Excel spreadsheet, consists of a series of subroutines designed to automatically measure rAd vector objects from the FESEM images. The application and utility of this macro program has enabled us to rapidly and efficiently analyze very large data sets of rAd samples while minimizing operator time.     

Enantiomeric resolution of underivatized small peptides by HPLC with a chiral crown ether stationary phase

Factors influencing the stereoisomeric resolution of underivatized dipeptides and a representative tripeptide on Crownpak (CR) columns have been investigated. The elution order and relative retention suggest that a combination of chiral, steric, and hydrophobic interactions effects the extent of chiral recognition and the retention achieved during separations. Some dipeptides whose amine terminus is located three atoms from the asymmetric center (such as dipeptides of D ,L -glycine) were resolved, but the elution order was the opposite of that expected for the type of Crownpak column used (CR(+)). Peptides containing hydrophobic substituents were strongly retained, but their retention times could be significantly reduced, and detectability improved, by use of gradient elution. Analysis of a commercial sample of D ,L -leucine-D ,L -alanine revealed the stereoisomers to be present in an unexpected quantitative ratio and demonstrated the utility of these separations for quality assurance and quantitative analyses.     

A short synthesis of C-glycosyl pyrazoles and pyrroles

The sodium salt of TOSMIC reacted with (E)-3,4,5,6,7-penta-O-acetyl-1,2-dideoxy-1-C-nitro-D-galacto- (1) and -D-manno-hept-1-enitol (2) to give 3-(D-galacto- (3) and 3-(D-manno-penta-O-acetylpentitol-1-yl)-4-nitropyrrole (4) , respectively. Compound 1 reacted with diaryl nitrile imines, affording 1,3-diaryl-4-(1,2,3,4,5-penta-O-acetyl-D-galacto-pentitol-1-yl)pyrazoles 5 and 6 .     

The Role of Multiple Teaching Strategies in Promoting Active Learning in Organic Chemistry

We have introduced four alternative teaching strategies into our yearlong organic chemistry course and have assessed changes in student performance relative to the same course taught by the same instructors using a more traditional lecture format. These strategies, which include reading worksheets, dialogues, in-class worksheets, and role-playing, allow the professor to move through a learning cycle that may effectively accommodate the students needs and multiple learning styles. The reading worksheets guide students through the concept phase and dialogues help students identify the importance of the concepts as they articulate these ideas for themselves. Group worksheets and role-playing provide opportunities for peer-interaction, application of knowledge, creativity, and self-expression. Others have shown that active learning strategies neither enhance nor diminish a students ability to retain factual information, a finding that is supported by our study. Our data from this one study show, however, that students taught by a more traditional approach demonstrate a greater variation in final exam performance between first and second semesters than those taught using a combination of techniques. This result reflects a shift in emphasis from the professor as an information source to the actively engaged student taking responsibility for his or her own learning. This study suggests that these methods, when used in a consistent fashion in conjunction with interactive lecturing, provide a broad base to facilitate student learning and aid in the development of higher order thinking skills.     

Development of the continuously variable volume reactor for flow injection analysis: Part 1. Design, capabilities and testing

A new apparatus for mixing sample and reagent in flow injection analysis (FIA) is described. The continuously variable volume reactor (CVVR) replaces the conventional mixing coil in a flow injection (FI) manifold to provide mixing and dilution. A linear actuator motor allows control of the chamber volume via LabVIEW software. The chamber volume can be incremented in steps of 1 μl over the range 68-1704 μl. In addition, the chamber has an integral variable-speed stirring unit that is also under computer control. Experiments were performed to evaluate the dispersion characteristics of this new device, evaluate the volume reproducibility, and understand the mixing characteristics. Use of the chamber is shown in the determination of iron(II) in pond water, and in NIST SRM 1643d with excellent results and a detection limit of 3.7 μg/l iron(II). Advantages of the CVVR and future research activities using the device are discussed.