Rapid Characterization of Combinatorial Libraries using Electrospray
Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.
Pp. 23-34.
A. S. Fang, P. Vouros, C. C. Stacey, G. H. Kruppa, F. H. Laukien,
F. A. Wintner, T. Carell and J. Rebek, Jr.
[Abstract]
Evaluation of the Antioxidant Potential of Natural Products. Pp. 35-46.
Sang Kook Lee, Zakaria H. Mbwambo, Ha-Sook Chung, Lumonadio Luyengi,
Esperanza J.C. Gamez, Rajendra G. Mehta, A. Douglas Kinghorn and John M.
Pezzuto
[Abstract]
Screening for Inhibitors of Dihydrofolate Reductase Using Pulsed Ultrafiltration
Mass Spectrometry. Pp. 47-55.
Dejan Nikolic and Richard B. van Breemen
[Abstract]
[Back to top] Rapid Characterization
of Combinatorial Libraries using Electrospray Ionization Fourier Transform
Ion Cyclotron Resonance Mass Spectrometry. A. S. Fang, P. Vouros, C. C.
Stacey, G. H. Kruppa, F. H. Laukien, F. A. Wintner, T. Carell and J. Rebek,
Jr.
The relatively new field of combinatorial chemistry has enabled researchers
to create large mixtures of compounds that can be screened for leads in
developing potential drug candidates. The new synthetic method has also
created a need for better procedures to analyze the complex mixtures that
are generated. The immediate goal in most cases is to verify the synthetic
procedure and to determine the purity and completeness of the library sample
before binding studies are initiated. We report here a method to rapidly
characterize small-molecule combinatorial libraries in solution. All combinatorial
library samples were synthesized by combining a core molecule bearing two
acid chloride functionalities with various amino acids to generate libraries
of 36, 78 and 120 components. Using electrospray ionization fourier transform
ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) we were able to
identify 70-80% of the library components. All samples were analyzed as
mixtures by direct infusion without chromatographic separation. Furthermore,
nominally isobaric components could be resolved and identified through
exact mass assignments without tandem mass spectrometery. ESI-FTICR-MS
is a rapid and convenient tool for the characterization of small-molecule
libraries. The method is especially useful for the analysis of larger libraries
that contain many nominally isobaric components and impurities.
[Back to top] Evaluation
of the Antioxidant Potential of Natural Products. Sang Kook Lee, Zakaria
H. Mbwambo, Ha-Sook Chung, Lumonadio Luyengi, Esperanza J.C. Gamez, Rajendra
G. Mehta, A. Douglas Kinghorn and John M. Pezzuto.
Since reactive oxygen radicals play an important role in carcinogenesis
and other human disease states, antioxidants present in consumable fruits,
vegetables, and beverages have received considerable attention as cancer
chemopreventive agents. Thus, in order to identify antioxidants in plant
extracts, test materials were assessed for potential to scavenge stable
1,2-diphenyl-2-picrylhydrazyl (DPPH) tree radicals, reduce TPA-induced
free radical formation in cultured HL-60 human leukemia cells, and inhibit
responses observed with a xanthine/xanthine oxidase assay system. Approximately
700 plant extracts were evaluated, and 28 were found to be active in the
DPPH free radical scavenging assay. Based on secondary analyses performed
to assess inhibition of 7,12-dimethylbenz(a)anthracene-induced preneoplastic
lesion formation with a mouse mammary organ culture model, Chorizanthe
diffusa Benth. (Polygonaceae), Mezoneuron cucullatum Roxb. (Leguminosae),
Cerbera manghas L. (Apocynaceae) and Daphniphyllum calycinum
Benth. (Daphniphyllaceae) were selected and subjected to bioassay-guided
fractionation. 5,7,3',5'-Tetrahydroxy-8,4'-dimethoxyflavonol, 5,8,4'-trihydroxy-7,3'-dimethoxyflavonol,
5,3',4'-trihydroxy-7-methoxyflavonol, and 6,3',4'-trihydroxy-7-methoxyflavonol
were identified as active principles from C. diffusa. Piceatannol,
trans-resveratrol, apigenin and scirpusin A were found as the active principles
of M. cucullatum, olivil, (-)-carinol, and (+)-cycloolivil were
active principles from C. manghas, and 5,6,7,4'-tetrahydroxyflavone
3-0-rutinoside and kaempterol 3-0-neohesperidoside were active principles
from D. calycinum. Of these substances, the hydroxystilbenes piceatannol
and trans-resveratrol have thus far been shown to inhibit carcinogen-induced
preneoplastic lesion formation in the mouse mammary gland organ culture
model.
[Back to top] Screening
for Inhibitors of Dihydrofolate Reductase Using Pulsed Ultrafiltration
Mass Spectrometry. Dejan Nikolic and Richard B. van Breemen.
A method of screening combinatorial libraries for inhibitors of eukaryotic
dihydrofolate reductase has been developed using pulsed ultra-filtration
electrospray mass spectrometry, which is a continuous-flow affinity separation
system for extracting and identifying high affinity ligands in combinatorial
libraries. In this application, pulsed ultrafiltration conditions were
optimized for the isolation and identification of inhibitors of dihydrofolate
reductase from a 22 compound library containing six known inhibitors of
the enzyme including trimethoprim, aminopterin, methotrexate, pyrimethamine,
folic acid, and folinic acid, and 16 compounds without known affinity.
In order to optimize the screening method, sources of non-specific binding
were identified and minimized. A significant source of non-specific binding
for this set of library compounds was hydrophobic interaction with the
surfaces of the ultrafiltration chamber. After affinity separation of bound
(high affinity) versus free (low affinity) library compounds during pulsed
ultrafiltration, receptor-bound ligands were released and eluted using
either organic solvent or acidified mobile phase. Although 80% methanol
easily disrupted the receptor-ligand complexes, organic solvent had the
undesirable effect of releasing non-specifically bound compounds from the
chamber and thereby increasing the background noise. Interference from
non-specific binding was minimized by releasing bound ligands using a low
pH mobile phase eluent instead of organic solvent. Under the conditions
used, pulsed ultrafiltration mass spectrometry selectively identified the
two library compounds with the highest affinity for dihydrofolate reductase,
methotrexate and aminopterin.