ABSTRACT
We investigated the separation and detection of the 5'-monophosphates of 2'-deoxynucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). BODIPY conjugates of the four common deoxynucleoside-5'-monophosphates (2'-deoxyguanosine-5'-monophosphate, 2'-deoxyadenosine-5'-monophosphate, 2'-deoxycytidine-5'-monophosphate, and thymidine-5'-monophosphate) were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions for the analysis of DNA. BODIPY conjugates were detected and resolved by CE-LIF after digestion of DNA or an oligonucleotide to 5'-monophosphates by nuclease P1 (NP 1) and fluorescence labeling without further purification step. Comparative analyses of calf-thymus DNA digested either with micrococcal nuclease/spleen phosphodiesterase to 3'-monophosphates or with NP 1 to 5'-monophosphates showed that both versions of the fluorescence postlabeling assay were equally efficient and sensitive. Moreover, using the same assay, 2'-deoxyuridine and 2'-deoxy-5methylcytidine were identified in bisulfite treated DNA after NP 1 digestion indicating that fluorescence postlabeling of 2'-deoxyribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF has the potential to determine DNA damage and genomic DNA methylation.
Subject(s)
Boron Compounds/chemistry , Deoxyribonucleotides/analysis , Deoxyribonucleotides/isolation & purification , Electrophoresis, Capillary/methods , Ethylenediamines/chemistry , Fluorescent Dyes/chemistry , DNA/drug effects , Deoxyadenine Nucleotides/isolation & purification , Deoxycytidine Monophosphate/isolation & purification , Deoxyguanine Nucleotides/isolation & purification , Lasers , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, Fluorescence , Sulfites/chemistry , Thymidine Monophosphate/isolation & purificationABSTRACT
Tumor targeting of glycoconjugated antineoplastic agents is a strategy currently under investigation for cancer chemotherapy. We have synthesized the glucosides and galactosides of the clinically established drug hydroxyurea and of mesylglycol, the reactive moiety of the anticancer drug busulfan. Glycosides of hydroxyurea were obtained by carbamoylation of hydroxylamine glycosides. The glycosides of mesylglycol were synthesized by mesylation of protected glycol glycosides. All compounds were characterized by detailed 1H and 13C NMR analysis.
Subject(s)
Antineoplastic Agents/chemical synthesis , Drug Delivery Systems/methods , Glycoconjugates/chemical synthesis , Hydroxyurea/analogs & derivatives , Hydroxyurea/chemistry , Mesylates/chemistry , Neoplasms/drug therapy , Antineoplastic Agents/chemistry , Glycoconjugates/chemistry , Magnetic Resonance Spectroscopy , Molecular StructureABSTRACT
The synthesis of a new ortho-carborane derivative, tetracarboranylketone 4, is reported here. Ketone 4 was prepared from a tetraalkynylated ketone by the addition of decaborane. The keto group was then easily modified to yield the glycosides 17alpha and 18beta, which contain glucose or galactose, respectively, and the nucleotide 13b. In addition to ketone 4, which is acyclic, cyclic ketone 8 was also synthesised. X-ray diffraction analysis of compound 4 indicated the presence of two toluene guest molecules per molecule of the host compound. Furthermore, compound 4 displays a rather low cytotoxicity. These novel products can be used as building blocks to create a new class of biomolecules containing high-density carborane clusters. Such molecules may constitute powerful tools for applications like Boron Neutron Capture Therapy or Energy-Filtering Transmission Electron Microscopy.
Subject(s)
Boron Compounds/chemistry , Boron Compounds/chemical synthesis , Boron Neutron Capture Therapy/methods , Boron/chemistry , Microscopy, Electron/methods , Pentanones/chemistry , Pentanones/chemical synthesis , Animals , Boron Compounds/toxicity , Cell Division/drug effects , Cell Line , Filtration , Glycosylation , Humans , Magnetic Resonance Spectroscopy , Molecular Conformation , Molecular Structure , Pentanones/toxicity , Rats , X-Ray DiffractionABSTRACT
An analytical method to determine the genome-wide DNA methylation in only 100 ng DNA is presented. The analysis is based on DNA isolation and hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with a fluorescence dye (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride, Bodipy FL EDA). The separation of the derivatives was carried out by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. To calculate the methylation level, the derivatization factor and the quantum yields of the Bodipy conjugates of 2'-desoxycytidine-3'-monophosphate (dCMP) and 2'-desoxy-5-methylcytidine-3'-monophosphate (5m-dCMP) were determined by measurement of methylated Lambda DNA. The assignment was made by cochromatography with the synthesized and characterized standard compound 5m-dCMP. After optimization of the method it was possible to determine the methylation level in 100-ng DNA samples with a standard deviation of less than 5%.
Subject(s)
DNA, Neoplasm/analysis , Deoxycytidine Monophosphate/analogs & derivatives , Electrophoresis, Capillary/methods , Fluorescent Dyes/chemistry , Lasers , Neoplasms/diagnosis , Bacteriophage lambda/chemistry , Bacteriophage lambda/genetics , Boron Compounds/chemistry , Breast Neoplasms/chemistry , Breast Neoplasms/genetics , Cell Line , DNA Methylation , Deoxycytidine Monophosphate/analysis , Humans , Neoplasms/chemistry , Neoplasms/geneticsABSTRACT
Increased excretion of ethylated DNA bases has been reported in the urine of cigarette smokers. To study DNA ethylation in the target organs of smokers, an immunoenriched (32)P-postlabeling assay for O(4)-ethylthymidine (O(4)-etT) was developed. O(4)-etT-3'-monophosphate (O(4)-etT-3'P) was synthesized, purified, and characterized by LC-MS, ESI-MS, and NMR. DNA was enzymatically digested to 2'-deoxynucleoside-3'-monophosphate followed by immunoprecipitation of O(4)-etT-3'P using specific monoclonal antibodies. The immunoconjugate was washed by filtration, and O(4)-etT-3'P was recovered by ethanol treatment. The enriched O(4)-etT-3'P was labeled with [gamma-(32)P]ATP in the presence of T4-polynucleotide kinase at pH 6.8 to yield its 5'-labeled monophosphate and was subsequently resolved on RP-HPLC and detected with online detection of radioactivity. Adduct recovery was >80%, and the detection limit was approximately 500 amol. To further validate the method, O(4)-etT levels were determined in calf thymus DNA treated with N-ethyl-N-nitrosourea, and a dose-dependent formation of O(4)-etT was observed. Furthermore, O(4)-etT was found to be present in the cells obtained from the lower respiratory tract by sputum induction of two out of four smokers but not in three nonsmokers. O(4)-etT is a poorly repaired promutagenic DNA lesion; thus, it could be of potential use for biomonitoring smoking-related DNA damage. Our improved assay was found to be sufficiently sensitive and specific to detect O(4)-etT in surrogate cells from cigarette smoke exposed humans.
