Biomedical subjects
Publications and source records attributed to T Carell.
Structural analysis, biochemistry and model studies have provided new insights into the mechanism of action of photolyases. The light-driven electron and energy transfer events that lead to the photolyase-catalyzed repair of lethal, mutagenic and carcinogenic UV-light-induced DNA lesions have all been examined in the past few years.
[reaction--see text] The synthesis of a vitamin B(2)-derived flavin-nucleotide is described. A combined H-phosphonate/phosphoramidite protocol was developed for the first incorporation of flavin coenzymes into a DNA stack. The coenzyme-DNA is predicted to have novel biosensing and catalytic properties.
Cyclobutane uridine and thymidine dimers with cis-syn-structure are DNA lesions, which are efficiently repaired in many species by DNA photolyases. The essential step of the repair reaction is a light driven electron transfer from a reduced FAD cofactor (FADH ) to the dimer lesion, which splits spontaneously into the monomers. Repair studies with UV-light damaged DNA revealed significant rate differences for the various dimer lesions. In particular the effect of the almost eclipsed positioned methyl groups at the thymidine cyclobutane dimer moiety on the splitting rates is unknown. In order to investigate the cleavage vulnerability of thymine and uracil cyclobutane photodimers outside the protein environment, two model compounds, containing a thymine or a uracil dimer and a covalently connected flavin, were prepared and comparatively investigated. Cleavage investigations under internal competition conditions revealed, in contrast to all previous findings, faster repair of the sterically less encumbered uracil dimer. Stereoelectronic effects are offered as a possible explanation. Ab initio calculations and X-ray crystal structure data reveal a different cyclobutane ring pucker of the uracil dimer, which leads to a better overlap of the pi*-C(4)-O(4)-orbital with the sigma*-C(5)-C(5')-orbital. Enzymatic studies with a DNA photolyase (A. nidulans) and oligonucleotides, which contain either a uridine or a thymidine dimer analogue, showed comparable repair efficiencies for both dimer lesions. Under internal competition conditions significantly faster repair of uridine dimers is observed.
The major UV-B photoproduct in DNA is the cyclobutane pyrimidine dimer (CPD). CPD-photolyases repair this DNA damage by a light-driven electron transfer. The chromophores of the class II CPD-photolyase from Arabidopsis thaliana, which was cloned recently [Taylor, R., Tobin, A. & Bray, C. (1996) Plant Physiol. 112, 862; Ahmad, M., Jarillo, J.A., Klimczak, L.J., Landry, L.G., Peng, T., Last, R.L. & Cashmore, A.R. (1997) Plant Cell 9, 199-207], have not been characterized so far. Here we report on the overexpression of the Arabidopsis CPD photolyase in Escherichia coli as a 6 x His-tag fusion protein, its purification and the analysis of the chromophore composition and enzymatic activity. Like class I photolyase, the Arabidopsis enzyme contains FAD but a second chromophore was not detectable. Despite the lack of a second chromophore the purified enzyme has photoreactivating activity.
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.
By means of capillary electrophoresis coupled online to electrospray ionization MS, a library of theoretically 171 disubstituted xanthene derivatives was analyzed. The method allowed the purity and makeup of the library to be determined: 160 of the expected compounds were found to be present, and 12 side-products were also detected in the mixture. Due to the ability of capillary electrophoresis to separate analytes on the basis of charge, most of the xanthene derivatives could be resolved by simple capillary electrophoresis-MS procedures even though 124 of the 171 theoretical compounds were isobaric with at least one other molecule in the mixture. Any remaining unresolved peaks were resolved by MS/MS experiments. The method shows promise for the analysis of small combinatorial libraries with fewer than 1000 components.
The growing interest in combinatorial chemistry has led us to explore new analytical methods for the analysis of complex molecular libraries. Because an investigation of large mixtures with 10(4)-10(5) different chemical entities was not realistic, an alternative approach was pursued that included the analysis of small representative sublibraries using positive and negative ion electrospray mass spectrometry. The detailed analysis of these model mixtures, containing up to 55 components, allowed us to obtain important information about the composition of a library with considerable complexity. The results were used to improve the synthetic procedure in order to provide the maximum yield of expected library components. The applicability of mass spectrometry to the analysis of complex matrices and the usefulness of the technique for screening synthesized combinatorial libraries to probe their expected diversity and complexity have been demonstrated.
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.