Yuriy M. Dunayevskiy

New promise in combinatorial chemistry: synthesis, characterization, and screening of small-molecule libraries in solution

BACKGROUND The increasing interest in combinatorial chemistry as a tool for the development of therapeutics has led to many new methods of creating molecular libraries of potential lead compounds. Current methods have made it possible to develop libraries of several million compounds. As a result, the limiting factor in the screening of libraries has become the identification and characterization of active species. We have recently described a method for generating libraries of water-soluble compounds containing mixtures of 10(4) to 10(5) different small organic molecules by using generally applicable solution phase chemistry. We set out to develop new methods to characterize and decode these libraries. RESULTS Libraries were generated by condensing a multi-acid-chloride core molecule with various amines, producing molecules with functional groups about a rigid backbone. Composition and complexity of the libraries was evaluated using electrospray mass spectrometry to analyze model libraries containing up to 55 different molecules. The number of peaks obtained in mass spectrometry is directly correlated with the complexity of the library, and we were therefore able to deduce which of the expected compounds had in fact been formed in the library, and which of the building blocks in the library were not efficiently used. An iterative selection procedure was developed using this information, which allowed the screening of libraries of up to 50,000 chemical species to produce a competitive inhibitor of the enzyme trypsin. CONCLUSIONS Our strategy for the identification of active species should be broadly applicable to other methods of generating complex libraries of small molecules. The selection from the library of a compound with desired biological properties augurs well for the potential value of generating and screening complex mixtures of small molecules in solution.

INTEGRATED MULTICHANNEL MICROCHIP ELECTROSPRAY IONIZATION MASS SPECTROMETRY : ANALYSIS OF PEPTIDES FROM ON-CHIP TRYPTIC DIGESTION OF MELITTIN

In continuation of our work to develop an integrated multichannel microchip interfaced to electrospray mass spectrometry (ESI-MS), this paper demonstrates one of several applications of this approach in monitoring tryptic digestion products. The multichannel microchip allowed integration of sample preparation onto the microchip to facilitate the analysis process. Melittin was selected as a model oligopeptide because it possesses a cluster of four adjacent basic residues which enable probing the site specificity of trypsin as a function of digest times. Reactions were performed on-chip in different wells for specific time periods and then analyzed by infusion from the microchip by ESI-MS, using leucine enkephalin as internal standard. The rate of formation and disappearance of the molecular ion and individual fragments was followed for a melittin to trypsin concentration ratio of 300:1. The results indicate the potential of integrating enzymatic reactions with multichannel microchip ESI-MS for automated optimization of reaction condition while consuming only small amounts of sample.

Mass spectrometric identification of ligands selected from combinatorial libraries using gel filtration

There is a constant search for a successful analytical methodology to provide high throughput screening of combinatorial libraries against biological targets for identification of active ligands. Solid-phase screening assays offer faster isolation and identification of active analytes compared to the solution-based iterative methods. On the other hand, shift of combinatorial research to the creation of soluble non-peptide libraries, and limitations associated with the heterogeneous assays, creates a demand for a breakthrough technology for rapid and efficient screening of combinatorial libraries in solution. We demonstrated the efficient and rapid approach for selecting active ligands from a combinatorial mixture with subsequent identification of compounds by mass spectrometry. The procedure involves the use of a biological target molecule to physically isolate the active component in a mixture on a size exclusion medium. Then the ligands are identified using a combined liquid chromatography/capillary electrophoresis/mass spectrometry system. As a model system we used serum albumin and small molecules with different affinities to the protein.

On the survivability and pyrosynthesis of PAH during combustion of pulverized coal and tire crumb

Abstract Results are presented on the emissions of semivolatile polycyclic aromatic hydrocarbons (PAH) from the combustion of a pulverized bituminous coal and ground waste automobile tires. Streams of fuel particles were injected at steady-state steady-flow conditions, and burned inside an isothermal drop-tube furnace, in air, at a gas temperature and gas residence time of 1150°C and 0.75 s, respectively. Combustion occurred under either very fuel-lean conditions (bulk equivalence ratio, φ 1.6 and, especially, under pyrolytic conditions in N 2 . These PAHs were mostly attributed to pyrosynthesis since none of the deuterated PAHs, adsorbed on the fuels, survived the combustion process. Small amounts of the labeled compounds, however, survived under purely pyrolytic conditions. These results were confirmed with separate experiments, where deuterium-labeled PAH standards were adsorbed on highly porous calcium/magnesium oxide or mullite particles. Again, small amounts of some PAHs survived in high-temperature pyrolytic conditions, but none in oxidative environments. These observations suggest that pyrosynthesis is the major contributing mechanism to the PAH emissions from the combustion of these fuels. Survivability of parent PAHs may be a minor mechanism at very high equivalence ratios. Finally, both fuels were mixed with powders of calcium magnesium acetate (CMA), calcium carbonate (CaCO 3 ), and calcium oxide (CaO), all of which are known sulfur reduction agents, at a molar Ca/S ratio of 1. Combustion of the fuels mixed with CMA or CaCO 3 generated enhanced amounts of PAHs, while combustion with CaO had no effect on the PAH emissions.

Aromatic Hydrocarbon Emissions from Burning Poly(styrene), Poly(ethylene) and PVC Particles at High Temperatures

A study on the semi-volatile aromatic hydrocarbon emissions from the pyrolysis/combustion of poly(styrene) (PS), poly(ethylene) (PE) and PVC particles was conducted. Dispersions (aerosols) or batches (fixed beds) of the above types of polymer particles, 90-300 μm in diameter, were burned in bench-scale, drop-tube or muffle-type electrically-heated furnaces, respectively. In the drop-tube furnace, pyrolysisis/combustion took place at gas temperatures ranging from 900 to 1200°C and particle heating rates were in the order of 10 3 -10 4 °C/s. The total residence times of the gases in the furnaces were 1 or 2 s ; envelope flames surrounding the particles lasted for 6-130 ms, while residence times in the post-flame region were 0.3-1.8 s depending on the polymer. Semi-volatile organic emissions were captured from the gas and solid phases using XAD-4 adsorbers and glass fiber filters, respectively, and analysis was conducted with gas chromatography coupled with mass spectrometry (GC-MS). The combustion characteristics of single particles of the three plastics were studied in earlier experiments using optical pyrometry and high-speed cinematography. Gaseous diffusion, chemical kinetics and pyrolysis kinetics were inferred, therein, to govern combustion of PS, PVC and PE particles, respectively. The types of the aromatic hydrocarbon emissions were found to depend on the polymer burned, while their relative amounts were influenced by combustion parameters such as temperature, residence time, etc. Substituted poly-aromatic hydrocarbons (PAHs), oxygenated compounds and chlorinated aromatics were detected in the combustion products of PVC. PAHs with fused rings were emitted from the combustion of PE. PAHs, either substituted or with fused rings were emitted from the combustion of PS. As the gas temperatures and residence times increased, the amount of PAH emissions from all polymers decreased. The results indicated that as the size of PVC sample decreased, in the presence of oxygen, the amount of PAHs decreased. However, as the size of PE sample decreased, or when oxygen was present, the amount of PAHs did not necessarily decrease. The behavior of PS was found to be between that of PVC and PS.