CCHTS Contents and Abstracts, Vol 2, No. 6

Volume 2, Number 6, 1999: Contents

Recent Advances in Liquid-Phase Combinatorial Chemistry. Pp. 299-318.
Chung-Ming Sun
[Abstract]

Generation of a Polyclonal Fab Phage Display Library to the Protozoan Parasite Cryptosporidium parvum. Pp. 319-325.
C. M. Baecher-Allan, K. Santora, S. Sarantopoulos, W. Den, S. R. Sompuram, A. M. Cevallos, N. Bhat, H. Ward and J. Sharon
[Abstract]

A Multiple Electrospray Interface for Parallel Mass Spectrometric Analyses of Compound Libraries. Pp. 327-334.
T. Wang, L. Zeng, J. Cohen and Daniel B. Kassel
[Abstract]

Parallel Solution Synthesis of a "Carbohybrid" Library Designed to Inhibit Galactose-Binding Proteins. Pp. 335-352.
U.J. Nilsson, E.J.-L. Fournier, E.J. Fryz and O. Hindsgaul
[Abstract]

Assays of Ligand-Human Serum Albumin Binding Using Pulsed Ultrafiltration and Liquid Chromatography-Mass Spectrometry. Pp. 353-359.
Chungang Gu, Dejan Nikolic, Jacob Lai, Xiaoying Xu and Richard B. van Breemen
[Abstract]

Abstracts

[Back to top] Recent Advances in Liquid-Phase Combinatorial Chemistry. Chung-Ming Sun.
In combination with high throughput screening, combinatorial organic synthesis of large numbers of pharmaceutically interesting compounds may revolutionize the drug discovery process. Although combinatorial organic synthesis on solid supports is a useful approach, several groups are focusing their research efforts on liquid-phase combinatorial synthesis by the use of soluble polymer supports to generate libraries. This macromolecular carrier, in contrast to an insoluble matrix, is soluble in most organic solvents and has a strong tendency for precipitation in particular solvents. Liquid-phase combinatorial synthesis is a unique approach since homogeneous reaction conditions can be applied, but product purification similar to the solid-phase method can be carried out by simple filtration and washing. This method combines the positive aspects of classical solution-phase chemistry and solid-phase synthesis. This review examines the recent applications (1995-1999) of soluble polymer supports in the synthesis of combinatorial libraries.

[Back to top] Generation of a Polyclonal Fab Phage Display Library to the Protozoan Parasite Cryptosporidium parvum. C. M. Baecher-Allan, K. Santora, S. Sarantopoulos, W. Den, S. R. Sompuram, A. M. Cevallos, N. Bhat, H. Ward and J. Sharon.
We had developed a technology for creation of recombinant polyclonal antibody libraries, standardized perpetual mixtures of polyclonal whole antibodies for which the genes are available and can be altered as desired. We report here the first phase of generating a polyclonal antibody library to Cryptosporidium parvum, a protozoan parasite that causes severe disease in AIDS patients, for which there is no effective treatment. BALB/c mice, immunized by neonatal oral infection with oocysts followed by intraperitoneal immunization with a sporozoite/oocyst preparation of C. parvum, were used for construction of a Fab phage display library in a specially-designed bidirectional vector. This library was selected for reactivity to an oocyst/sporozoite preparation, and was shown to be antigen-specific and diverse. Following mass transfer of the selected variable region gene pairs to appropriate mammalian expression vectors, such anti-C. parvum Fab phage display libraries could be used to develop chimeric polyclonal antibody libraries, with mouse variable regions and human constant regions, for passive immunotherapy of C. parvum infection.

[Back to top] A Multiple Electrospray Interface for Parallel Mass Spectrometric Analyses of Compound Libraries. T. Wang, L. Zeng, J. Cohen and Daniel B. Kassel.
A parallel spray interface for mass spectrometry is described. This new electrospray interface enables effluent flow streams from an array of HPLC columns to be sampled independently and sequentially on a chromatographic time-scale. Unlike our previously reported parallel LC-MS interface, which incorporated a dual-sheath spray interface accommodating up to four flow streams that are sampled simultaneously, this new interface permits up to four columns to be sampled sequentially by means of a stepping motor and rotating plate assembly. Effluent flow streams from an array of four HPLC columns are connected to a parallel arrangement of electrospray needles co-axial to the mass spectrometer entrance aperture. Within the needle assembly, the individual spray tips are oriented in a circular array, where each needle is situated 90 degrees relative to one another for four-column operation. An eight-column system is described with needles spaced at 45 degree intervals. In between the needle assembly and the mass spectrometer entrance aperture is a Teflon disk with a through-hole that is mounted to a stepping motor assembly. By precisely controlling the stepping of the motor assembly, it is possible to "sample" each of the spray positions multiple times per second. By operating the quadrupole mass spectrometer in the single ion monitoring (SIM) mode, it was possible to acquire data at each of the spray positions during the course of the elution of compounds from the HPLC column array while maintaining both good sensitivity and peak shape. Preliminary results suggest this technique will be useful for high throughput combinatorial library analysis and profiling.

[Back to top] Parallel Solution Synthesis of a "Carbohybrid" Library Designed to Inhibit Galactose-Binding Proteins. U.J. Nilsson, E.J.-L. Fournier, E.J. Fryz and O. Hindsgaul.
Parallel solution S-alkylations of a 1-thio- b -D-galactopyranoside derivative with Michael acceptors and a -chloroketones, followed by ketone reductions, reductive aminations, and acylations were developed to yield a library of 1-thio- b -D-galactopyranosides carrying small and diverse polar-neutral, hydrophobic, aromatic, cationic, or anionic non-carbohydrate aglycon structures. Screening of the library against a panel of galactose recognizing plant lectins revealed mM inhibitors of toxin A of A. precatorius superior to the reference ligands lactose and N-acetyl lactosamine. Such small, monosaccharide based inhibitors are attractive lead-molecules for therapeutic development, since they are low-molecular, hydrolytically stable and more hydrophobic than natural oligosaccharides.

[Back to top] Assays of Ligand-Human Serum Albumin Binding Using Pulsed Ultrafiltration and Liquid Chromatography-Mass Spectrometry. Chungang Gu, Dejan Nikolic, Jacob Lai, Xiaoying Xu and Richard B. van Breemen.
Two approaches were utilized to increase the throughput of pulsed ultrafiltration assays of ligand binding to human serum albumin, reducing the volume of the ultrafiltration chamber and combining pulsed ultrafiltration with high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-MS). Affinity constants for binding of ligands to human serum albumin were determined using pulsed ultrafiltration with ultraviolet absorbance detection. The first affinity constants (K_a1) were measured for the binding of dansylsarcosine, dansylamide, 7-anilinocoumarin-4-acetic acid and warfarin, and were determined to be 1.8 ¥ 10⁵, 5 ¥ 10⁴, 8 ¥ 10⁴, and 2.0 ¥ 10⁵ M^-1, respectively. The throughput of pulsed ultrafiltration analyses was tripled compared to previous pulsed ultrafiltration measurements by reducing the volume of the chamber. In addition, the use of LC-MS with pulsed ultrafiltration permitted the simultaneous comparison and rank ordering of ligand mixtures for binding to serum albumin. For example, the throughput of these pulsed ultrafiltration measurements was tripled by analyzing three ligands as a mixture.

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