Anthracyclines React with Apurinic/Apyrimidinic Sites in DNA.

The combination of doxorubicin (Adriamycin) and cyclophosphamide, referred to as AC chemotherapy, is commonly used for the clinical treatment of breast and other cancers. Both agents target DNA with cyclophosphamide causing alkylation damage and doxorubicin stabilizing the topoisomerase II-DNA complex. We hypothesize a new mechanism of action whereby both agents work in concert. DNA alkylating agents, such as nitrogen mustards, increase the number of apurinic/apyrimidinic (AP) sites through deglycosylation of labile alkylated bases. Herein, we demonstrate that anthracyclines with aldehyde-reactive primary and secondary amines form covalent Schiff base adducts with AP sites in a 12-mer DNA duplex, calf thymus DNA, and MDA-MB-231 human breast cancer cells treated with nor-nitrogen mustard and the anthracycline mitoxantrone. The anthracycline-AP site conjugates are characterized and quantified by mass spectrometry after NaB(CN)H3 or NaBH4 reduction of the Schiff base. If stable, the anthracycline-AP site conjugates represent bulky adducts that may block DNA replication and contribute to the cytotoxic mechanism of therapies involving combinations of anthracyclines and DNA alkylating agents.

Catalysts of DNA Strand Cleavage at Apurinic/Apyrimidinic Sites.

Apurinic/apyrimidinic (AP) sites are constantly formed in cellular DNA due to instability of the glycosidic bond, particularly at purines and various oxidized, alkylated, or otherwise damaged nucleobases. AP sites are also generated by DNA glycosylases that initiate DNA base excision repair. These lesions represent a significant block to DNA replication and are extremely mutagenic. Some DNA glycosylases possess AP lyase activities that nick the DNA strand at the deoxyribose moiety via a ß- or ß,δ-elimination reaction. Various amines can incise AP sites via a similar mechanism, but this non-enzymatic cleavage typically requires high reagent concentrations. Herein, we describe a new class of small molecules that function at low micromolar concentrations as both ß- and ß,δ-elimination catalysts at AP sites. Structure-activity relationships have established several characteristics that appear to be necessary for the formation of an iminium ion intermediate that self-catalyzes the elimination at the deoxyribose ring.

Characterization of nitrogen mustard formamidopyrimidine adduct formation of bis(2-chloroethyl)ethylamine with calf thymus DNA and a human mammary cancer cell line.

A robust, quantitative ultraperformance liquid chromatography ion trap multistage scanning mass spectrometric (UPLC/MS(3)) method was established to characterize and measure five guanine adducts formed by reaction of the chemotherapeutic nitrogen mustard (NM) bis(2-chloroethyl)ethylamine with calf thymus (CT) DNA. In addition to the known N7-guanine (NM-G) adduct and its cross-link (G-NM-G), the ring-opened formamidopyrimidine (FapyG) monoadduct (NM-FapyG) and cross-links in which one (FapyG-NM-G) or both (FapyG-NM-FapyG) guanines underwent ring-opening to FapyG units were identified. Authentic standards of all adducts were synthesized and characterized by NMR and mass spectrometry. These adducts were quantified in CT DNA treated with NM (1 µM) as their deglycosylated bases. A two-stage neutral thermal hydrolysis was developed to mitigate the artifactual formation of ring-opened FapyG adducts involving hydrolysis of the cationic adduct at 37 °C, followed by hydrolysis of the FapyG adducts at 95 °C. The limit of quantification values ranged between 0.3 and 1.6 adducts per 10(7) DNA bases when the equivalent of 5 µg of DNA hydrolysate was assayed on column. The principal adduct formed was the G-NM-G cross-link, followed by the NM-G monoadduct; the FapyG-NM-G cross-link adduct; and the FapyG-NM-FapyG was below the limit of detection. The NM-FapyG adducts were formed in CT DNA at a level ∼20% that of the NM-G adduct. NM-FapyG has not been previously quanitified, and the FapyG-NM-G and FapyG-NM-FapyG adducts have not been previously characterized. Our validated analytical method was then applied to measure DNA adduct formation in the MDA-MB-231 mammary tumor cell line exposed to NM (100 µM) for 24 h. The major adduct formed was NM-G (970 adducts per 10(7) bases), followed by G-NM-G (240 adducts per 10(7) bases), NM-FapyG (180 adducts per 10(7) bases), and, last, the FapyG-NM-G cross-link adduct (6.0 adducts per 10(7) bases). These lesions are expected to contribute to NM-mediated toxicity and genotoxicity in vivo.

Synthesis of G-N2-(CH2)3-N2-G Trimethylene DNA interstrand cross-links.

The synthesis of G-N2-(CH2)3-N2-G trimethylene DNA interstrand cross-links (ICLs) in a 5'-CG-3' and 5'-GC-3' sequence from oligodeoxynucleotides containing N2-(3-aminopropyl)-2'-deoxyguanosine and 2-fluoro-O6-(trimethylsilylethyl)inosine is presented. Automated solid-phase DNA synthesis was used for unmodified bases and modified nucleotides were incorporated via their corresponding phosphoramidite reagent by a manual coupling protocol. The preparation of the phosphoramidite reagents for incorporation of N2-(3-aminopropyl)-2'-deoxyguanosine is reported. The high-purity trimethylene DNA interstrand cross-link product is obtained through a nucleophilic aromatic substitution reaction between the N2-(3-aminopropyl)-2'-deoxyguanosine and 2-fluoro-O6-(trimethylsilylethyl)inosine containing oligodeoxynucleotides.

Synthesis of G-N2-(CH2)3-N2-G Trimethylene DNA Interstrand Cross-Links.

The synthesis of G-N(2)-(CH(2))(3)-N(2)-G trimethylene DNA interstrand cross-links (ICLs) in a 5'-CG-3' and 5'-GC-3' sequence from oligodeoxynucleotides containing N(2)-(3-aminopropyl)-2'-deoxyguanosine and 2-fluoro-O(6)-(trimethylsilylethyl)inosine is presented. Automated solid-phase DNA synthesis was used for unmodified bases and modified nucleotides were incorporated via their corresponding phosphoramidite reagent by a manual coupling protocol. The preparation of the phosphoramidite reagents for incorporation of N(2)-(3-aminopropyl)-2'-deoxyguanosine is reported. The high-purity trimethylene DNA interstrand cross-link product is obtained through a nucleophilic aromatic substitution reaction between the N(2)-(3-aminopropyl)-2'-deoxyguanosine- and 2-fluoro-O(6)-(trimethylsilylethyl)inosine-containing oligodeoxynucleotides.

Hyperpolarization of amino acid precursors to neurotransmitters with parahydrogen induced polarization.

Several important neurotransmitter precursors were hyperpolarized via homogeneous hydrogenation with parahydrogen. Polarization enhancement was achieved for (1)H and (13)C spins by several orders of magnitude compared to thermal spectra. Such large signal enhancements of these molecules could facilitate neurotransmitter studies.

Supramolecular metal displacement allows on-fluorescence analysis of manganese(II) in living cells.

Due to the importance of Mn(2+) ions in biological processes, it is of growing interest to develop protocols for analysis of Mn(2+) uptake and distribution in cells. A supramolecular metal displacement assay can provide ratiometric fluorescence detection of Mn(2+), allowing for quantitative and longitudinal analysis of Mn(2+) uptake in living cells.

Extended para-hydrogenation monitored by NMR spectroscopy.

A system that provides a sustained hyperpolarized (1)H NMR signal in an aqueous medium is reported. The enhanced signal lasts much longer than typical (1)H T(1) values, uncovering new possibilities for implementing hyperpolarized (1)H NMR/MRI experiments or performing kinetics studies that would not otherwise be detectable.