Synthesis of Macrocyclic Hexaoxazole (6OTD) Dimers, Containing Guanidine and Amine Functionalized Side Chains, and an Evaluation of Their Telomeric G4 Stabilizing Properties

Structure-activity relationship studies were carried out on macrocyclic hexaoxazole (6OTD) dimers, whose core structure stabilizes telomeric G-quadruplexes (G4). Two new 6OTD dimers having side chain amine and guanidine functional groups were synthesized and evaluated for their stabilizing ability against a telomeric G4 DNA sequence. The results show that the 6OTD dimers interact with the DNA to form 1:1 complexes and stabilize the antiparallel G4 structure of DNA in the presence of potassium cation. The guanidine functionalized dimer displays a potent stabilizing ability of the G4 structure, as determined by using a FRET melting assay (ΔTm = 14°C)

Intercalation-enhanced “Click” Crosslinking of DNA

DNA–DNA cross‐linking agents constitute an important family of chemotherapeutics that non‐specifically react with endogenous nucleophiles and therefore exhibit undesirable side effects. Here we report a cationic Sondheimer diyne derivative “DiMOC” that exhibits weak, reversible intercalation into duplex DNA (Kd=15 μm) where it undergoes tandem strain‐promoted cross‐linking of azide‐containing DNA to give DNA‐DNA interstrand crosslinks (ICLs) with an exceptionally high apparent rate constant kapp=2.1×105 m−1 s−1. This represents a 21 000‐fold rate enhancement as compared the reaction between DIMOC and 5‐(azidomethyl)‐2′‐deoxyuridine (AmdU) nucleoside. As single agents, 5′‐bispivaloyloxymethyl (POM)‐AmdU and DiMOC exhibited low cytotoxicity, but highly toxic DNA‐DNA ICLs were generated by metabolic incorporation of AmdU groups into cellular DNA, followed by treatment of the cells with DiMOC. These results provide the first examples of intercalation‐enhanced bioorthogonal chemical reactions on DNA, and furthermore, the first strain‐promoted double click (SPDC) reactions inside of living cells

In Vivo Incorporation of Azide Groups into DNA by Using Membrane-Permeable Nucleotide Triesters

Metabolic incorporation of bioorthogonal functional groups into cellular nucleic acids can be impeded by insufficient phosphorylation of nucleosides. Previous studies found that 5azidomethyl‐2′‐deoxyuridine (AmdU) was incorporated into the DNA of HeLa cells expressing a low‐fidelity thymidine kinase, but not by wild‐type HeLa cells. Here we report that membrane‐permeable phosphotriester derivatives of AmdU can exhibit enhanced incorporation into the DNA of wild‐type cells and animals. AmdU monophosphate derivatives bearing either 5′‐bispivaloyloxymethyl (POM), 5′‐bis‐(4‐acetoxybenzyl) (AB), or “Protide” protective groups were used to mask the phosphate group of AmdU prior to its entry into cells. The POM derivative “POM‐AmdU” exhibited better chemical stability, greater metabolic incorporation efficiency, and lower toxicity than “AB‐AmdU”. Remarkably, the addition of POM‐AmdU to the water of zebrafish larvae enabled the biosynthesis of azide‐modified DNA throughout the body

Synthesis of Deuterium-Labeled Vitamin D Metabolites as Internal Standards for LC-MS Analysis

Blood levels of the vitamin D3 (D3) metabolites 25-hydroxyvitamin D3 (25(OH)D3), 24R,25-dihydroxyvitamin D3, and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) are recognized indicators for the diagnosis of bone metabolism-related diseases, D3 deficiency-related diseases, and hypercalcemia, and are generally measured by liquid-chromatography tandem mass spectrometry (LC-MS/MS) using an isotope dilution method. However, other D3 metabolites, such as 20-hydroxyvitamin D3 and lactone D3, also show interesting biological activities and stable isotope-labeled derivatives are required for LC-MS/MS analysis of their concentrations in serum. Here, we describe a versatile synthesis of deuterium-labeled D3 metabolites using A-ring synthons containing three deuterium atoms. Deuterium-labeled 25(OH)D3 (2), 25(OH)D3-23,26-lactone (6), and 1,25(OH)2D3-23,26-lactone (7) were synthesized, and successfully applied as internal standards for the measurement of these compounds in pooled human serum. This is the first quantification of 1,25(OH)2D3-23,26-lactone (7) in human serum

Solution Structure of an Intramolecular (3 + 1) Human Telomeric G‑Quadruplex Bound to a Telomestatin Derivative

Guanine-rich human telomeric DNA can adopt secondary structures known as G-quadruplexes, which can be targeted by small molecules to achieve anticancer effects. So far, the structural information on complexes between human telomeric DNA and ligands is limited to the parallel G-quadruplex conformation, despite the high structural polymorphism of human telomeric G-quadruplexes. No structure has been yet resolved for the complex with telomestatin, one of the most promising G-quadruplex-targeting anticancer drug candidates. Here we present the first high-resolution structure of the complex between an intramolecular (3 + 1) human telomeric G-quadruplex and a telomestatin derivative, the macrocyclic hexaoxazole L2H2-6M(2)­OTD. This compound is observed to interact with the G-quadruplex through π-stacking and electrostatic interactions. This structural information provides a platform for the design of topology-specific G-quadruplex-targeting compounds and is valuable for the development of new potent anticancer drugs

A Novel Glucosylation Reaction on Anthocyanins Catalyzed by Acyl-Glucose–Dependent Glucosyltransferase in the Petals of Carnation and Delphinium[C][W]

This work describes a glucosylation reaction at the 5/7 positions of anthocyanins in the petals of carnations and delphiniums. Unusually, this reaction is catalyzed by acyl-glucose–dependent glucosyltransferases that belong to glycoside hydrolase family 1. This modification mechanism may play an important role in generating variation in anthocyanins

Disabling c-Myc in Childhood Medulloblastoma and Atypical Teratoid/Rhabdoid Tumor Cells by the Potent G-Quadruplex Interactive Agent S2T1-6OTD

We investigated here the effects of S2T1-6OTD, a novel telomestatin derivative that is synthesized to target G-quadruplex–forming DNA sequences, on a representative panel of human medulloblastoma (MB) and atypical teratoid/rhabdoid (AT/RT) childhood brain cancer cell lines. S2T1-6OTD proved to be a potent c-Myc inhibitor through its high-affinity physical interaction with the G-quadruplex structure in the c-Myc promoter. Treatment with S2T1-6OTD reduced the mRNA and protein expressions of c-Myc and hTERT, which is transcriptionally regulated by c-Myc, and decreased the activities of both genes. In remarkable contrast to control cells, short-term (72-hour) treatment with S2T1-6OTD resulted in a doseand time-dependent antiproliferative effect in all MB and AT/RT brain tumor cell lines tested (IC50, 0.25– 0.39 μmol/L). Under conditions where inhibition of both proliferation and c-Myc activity was observed, S2T1-6OTD treatment decreased the protein expression of the cell cycle activator cyclin-dependent kinase 2 and induced cell cycle arrest. Long-term treatment (5 weeks) with nontoxic concentrations of S2T1- 6OTD resulted in a time-dependent (mainly c-Myc–dependent) telomere shortening. This was accompanied by cell growth arrest starting on day 28 followed by cell senescence and induction of apoptosis on day 35 in all of the five cell lines investigated. On in vivo animal testing, S2T1-6OTD may well represent a novel therapeutic strategy for childhood brain tumors