Photoacoustic imaging of elevated glutathione in models of lung cancer for companion diagnostic applications.

Companion diagnostics (CDx) are powerful tests that can provide physicians with crucial biomarker information that can improve treatment outcomes by matching therapies to patients. Here, we report a photoacoustic imaging-based CDx (PACDx) for the selective detection of elevated glutathione (GSH) in a lung cancer model. GSH is abundant in most cells, so we adopted a physical organic chemistry approach to precisely tune the reactivity to distinguish between normal and pathological states. To evaluate the efficacy of PACDx in vivo, we designed a blind study where photoacoustic imaging was used to identify mice bearing lung xenografts. We also employed PACDx in orthotopic lung cancer and liver metastasis models to image GSH. In addition, we designed a matching prodrug, PARx, that uses the same SNAr chemistry to release a chemotherapeutic with an integrated PA readout. Studies demonstrate that PARx can inhibit tumour growth without off-target toxicity in a lung cancer xenograft model.

5-Aza-4'-thio-2'-deoxycytidine, a New Orally Bioavailable Nontoxic "Best-in-Class": DNA Methyltransferase 1-Depleting Agent in Clinical Development.

DNA methyltransferase (DNMT) 1 is an enzyme that functions as a maintenance methyltransferase during DNA replication, and depletion of this enzyme from cells is considered to be a rational goal in DNA methylation-dependent disorders. Two DNMT1-depleting agents 5-aza-2'-deoxycytidine (aza-dCyd, decitabine) and 5-aza-cytidine (aza-Cyd, azacitidine) are currently used for the treatment of myelodysplastic syndromes and acute myeloid leukemia and have also been investigated for nononcology indications, such as sickle cell disease. However, these agents have several off-target activities leading to significant toxicities that limit dosing and duration of treatment. Development of more selective inhibitors of DNMT1 could therefore afford treatment of long durations at effective doses. We have discovered that 5-aza-4'-thio-2'-deoxycytidine (aza-T-dCyd) is as effective as aza-dCyd in depleting DNMT1 in mouse tumor models but with markedly low toxicity. In this review we describe the preclinical studies that led to the development of aza-T-dCyd as a superior DNMT1-depleting agent with respect to aza-dCyd and will describe its pharmacology, metabolism, and mechanism of action. In an effort to understand why aza-T-dCyd is a more selective DNMT1 depleting agent than aza-dCyd, we will also compare and contrast the activities of these two agents. SIGNIFICANCE STATEMENT: Aza-T-dCyd is a potent DNMT1-depleting agent. Although similar in structure to decitabine (aza-dCyd), its metabolism and mechanism of action is different than that of aza-dCyd, resulting in less off-target activity and less toxicity. The larger therapeutic index of aza-T-dCyd (DNMT1 depletion vs. toxicity) in mice suggests that it would be a better clinical candidate to selectively deplete DNMT1 from target cells and determine whether or not depletion of DNMT1 is an effective target for various diseases.

A Liposomal Gemcitabine, FF-10832, Improves Plasma Stability, Tumor Targeting, and Antitumor Efficacy of Gemcitabine in Pancreatic Cancer Xenograft Models.

PURPOSE: The clinical application of gemcitabine (GEM) is limited by its pharmacokinetic properties. The aim of this study was to characterize the stability in circulating plasma, tumor targeting, and payload release of liposome-encapsulated GEM, FF-10832. METHODS: Antitumor activity was assessed in xenograft mouse models of human pancreatic cancer. The pharmacokinetics of GEM and its active metabolite dFdCTP were also evaluated. RESULTS: In mice with Capan-1 tumors, the dose-normalized areas under the curve (AUCs) after FF-10832 administration in plasma and tumor were 672 and 1047 times higher, respectively, than after using unencapsulated GEM. The tumor-to-bone marrow AUC ratio of dFdCTP was approximately eight times higher after FF-10832 administration than after GEM administration. These results indicated that liposomal encapsulation produced long-term stability in circulating plasma and tumor-selective targeting of GEM. In mice with Capan-1, SUIT-2, and BxPC-3 tumors, FF-10832 had better antitumor activity and tolerability than GEM. Internalization of FF-10832 in tumor-associated macrophages (TAMs) was revealed by flow cytometry and confocal laser scanning microscopy, and GEM was efficiently released from isolated macrophages of mice treated with FF-10832. These results suggest that TAMs are one of the potential reservoirs of GEM in tumors. CONCLUSION: This study found that FF-10832 had favorable pharmacokinetic properties. The liposomal formulation was more effective and tolerable than unencapsulated GEM in mouse xenograft tumor models. Hence, FF-10832 is a promising candidate for the treatment of pancreatic cancer.

Ultrasound-Mediated Gemcitabine Delivery Reduces the Normal-Tissue Toxicity of Chemoradiation Therapy in a Muscle-Invasive Bladder Cancer Model.

PURPOSE: Chemoradiation therapy is the standard of care in muscle-invasive bladder cancer (MIBC). Although agents such as gemcitabine can enhance tumor radiosensitivity, their side effects can limit patient eligibility and treatment efficacy. This study investigates ultrasound and microbubbles for targeting gemcitabine delivery to reduce normal-tissue toxicity in a murine orthotopic MIBC model. MATERIALS AND METHODS: CD1-nude mice were injected orthotopically with RT112 bladder tumor cells. Conventional chemoradiation involved injecting gemcitabine (10 mg/kg) before 6 Gy targeted irradiation of the bladder area using the Small Animal Radiation Research Platform (SARRP). Ultrasound-mediated gemcitabine delivery (10 mg/kg gemcitabine) involved either coadministration of microbubbles with gemcitabine or conjugating gemcitabine onto microbubbles followed by exposure to ultrasound (1.1 MHz center frequency, 1 MPa peak negative pressure, 1% duty cycle, and 0.5 Hz pulse repetition frequency) before SARRP irradiation. The effect of ultrasound and microbubbles alone was also tested. Tumor volumes were measured by 3D ultrasound imaging. Acute normal-tissue toxicity from 12 Gy to the lower bowel area was assessed using an intestinal crypt assay in mice culled 3.75 days posttreatment. RESULTS: A significant delay in tumor growth was observed with conventional chemoradiation therapy and both microbubble groups (P < .05 compared with the radiation-only group). Transient weight loss was seen in the microbubble groups, which resolved within 10 days posttreatment. A positive correlation was found between weight loss on day 3 posttreatment and tumor growth delay (P < .05; R2 = 0.76). In contrast with conventional chemoradiation therapy, ultrasound-mediated drug delivery methods did not exacerbate the acute intestinal toxicity using the crypt assay. CONCLUSIONS: Ultrasound and microbubbles offer a promising new approach for improving chemoradiation therapy for muscle-invasive bladder cancer, maintaining a delay in tumor growth but with reduced acute intestinal toxicity compared with conventional chemoradiation therapy.

Design, synthesis, and evaluation of liver-specific gemcitabine prodrugs for potential treatment of hepatitis C virus infection and hepatocellular carcinoma.

Many successful anti-viral and anti-cancer drugs are nucleoside analogs, which disrupt RNA and/or DNA synthesis. Here, we present liver-specific prodrugs of the chemotherapy drug gemcitabine (2',2'-difluorodeoxycytidine) for the treatment of hepatitis C virus (HCV) infection and hepatocellular carcinoma. The prodrugs were synthesized by introducing aromatic functional moieties to the cytosine 4-NH2 group of gemcitabine via amide bonds. The chemical modification was designed to i) enable passive diffusion across cellular membrane, ii) protect the prodrugs from inactivating deamination by cellular enzymes, and iii) allow release of active gemcitabine after amide hydrolysis by high levels of carboxylesterases in the liver. We found that many of our prodrugs exhibited similar toxicity as gemcitabine toward liver- and kidney-derived cancer cell lines but were 24- to 620-fold less cytotoxic than gemcitabine in breast- and pancreas-derived cancer cells, respectively. The prodrugs also inhibited an HCV replicon with IC50 values ranging from 10 nM-1.7 µM. Moreover, many of the prodrugs had therapeutic index values of >10,000 and have synergetic effects when combined with other Food and Drug Administration-approved anti-HCV small molecule drugs. These characteristics support the development of gemcitabine prodrugs as liver-specific therapeutics.

The development of ß-selective glycosylation reactions with benzyl substituted 2-deoxy-1,4-dithio-D-erythro-pentofuranosides: enabling practical multi-gram syntheses of 4'-Thio-2'-deoxycytidine (T-dCyd) and 5-aza-4'-thio-2'-deoxycytidine (aza-T-dCyd) to support clinical development.

The lack of effective methods to perform direct ß-selective glycosylation reactions with 2-deoxy-1,4-dithio-D-erythro-pentofuranosides has long been a significant stumbling block for the multi-gram synthesis of 4'-thio-2'-deoxy nucleosides. In addition, previously reported methods for the preparation of appropriately substituted 2-deoxy-1,4-dithio-D-erythro-pentofuranosides have proven problematic for large scale synthesis. To address these issues, herein we describe the modification and optimization of previously reported methods to allow for the convenient large scale synthesis of benzyl substituted 2-deoxy-1,4-dithio-D-erythro-pentofuranosides. Furthermore, we describe the development of reaction conditions for ß-selective glycosylation reactions of benzyl substituted 2-deoxy-1,4-dithio-D-erythro-pentofuranosides with both N4-benzoylcytosine and 5-aza-cytosine to enable the practical multi-gram syntheses of the clinical candidates 4'-thio-2'-deoxycytidine (T-dCyd) and 5-aza-4'-thio-2'-deoxycytidine (aza-T-dCyd). Taken together, these new synthetic developments have made possible the preclinical and early clinical development of these important anticancer agents at the National Cancer Institute.

Enhancement of Pancreatic Cancer Therapy Efficacy by Type-1 Matrix Metalloproteinase-Functionalized Nanoparticles for the Selective Delivery of Gemcitabine and Erlotinib.

PURPOSE: Pancreatic cancer (PCa) is projected to become the second leading cause of cancer-related deaths by 2030. Gemcitabine (GEM) combined with erlotinib (ERL) have been approved by the FDA for locally advanced, unresectable or metastatic pancreatic cancer therapy since 2005. Type-1 matrix metalloproteinase (MT1-MMP) has been recognized as a critical mediator of several steps in PCa progression including activating TGF-ß or releasing latent TGF-ß from LTBP-1, resulting in increased collagen production and cleavage collagen. METHODS: In the present research, GEM and ERL co-loaded nanoparticles (GEM/ERL NPs) were prepared. A non-substrate MT1-MMP binding peptide was decorated onto the GEM/ERL NPs surface. RESULTS: M-M GEM/ERL NPs exhibited the highest uptake ability (67.65 ± 2.87%), longest half-life period, largest area under the curve, and the best tumor inhibition efficiency (69.81 ± 4.13%). The body weight, blood urine nitrogen (BUN), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) of the system were steady when tested in mice model. CONCLUSION: In conclusion, M-M GEM/ERL NPs protected the drugs in the plasma, improved cellular uptake capacity, exhibited the most remarkable tumor cell inhibition ability, and showed the most efficient tumor growth inhibition capacity in vivo. M-M GEM/ERL NPs could be applied as an efficient and safe system for the synergistic combination chemotherapy of PCa.

pH and singlet oxygen dual-responsive GEM prodrug micelles for efficient combination therapy of chemotherapy and photodynamic therapy.

Nanocarriers have been an important strategy for enhancing the combination therapy of chemotherapy and photodynamic therapy (PDT) (Chem-PDT). However, conventional nanocarriers suffer from the problems of drug leakage in blood and insufficient drug release at target sites. Herein, we have designed a chlorin e6 (Ce6)-loaded GEM prodrug polymer micelle with a singlet oxygen cleavable linker and a pH responsive switch to avoid drug leakage in blood. These Ce6-loaded prodrug micelles possessed a uniform size distribution with a particle size of 78 nm. Meanwhile, the release of Ce6 and GEM was well controlled by acidic pH and laser irradiation. In addition, these micelles showed great acid triggered particle size shrinkage and charge-conversion properties, promoting micelle penetration at tumors and cellular uptake of micelles, which were confirmed by using CLSM of in vitro cell spheres and flow cytometry. Moreover, the in vitro and in vivo1O2 generation ability and antitumor ability of these micelles were impressive. This novel nanocarrier is a potential candidate for efficient Chem-PDT therapy.

Synthesis of 4'-Substituted-2'-Deoxy-2'-α-Fluoro Nucleoside Analogs as Potential Antiviral Agents.

Nucleoside analogs are widely used for the treatment of viral diseases (Hepatitis B/C, herpes and human immunodeficiency virus, HIV) and various malignancies. ALS-8176, a prodrug of the 4'-chloromethyl-2'-deoxy-2'-fluoro nucleoside ALS-8112, was evaluated in hospitalized infants for the treatment of respiratory syncytial virus (RSV), but was abandoned for unclear reasons. Based on the structure of ALS-8112, a series of novel 4'-modified-2'-deoxy-2'-fluoro nucleosides were synthesized. Newly prepared compounds were evaluated against RSV, but also against a panel of RNA viruses, including Dengue, West Nile, Chikungunya, and Zika viruses. Unfortunately, none of the compounds showed marked antiviral activity against these viruses.

Theranostic nanoparticles enabling the release of phosphorylated gemcitabine for advanced pancreatic cancer therapy.

Gemcitabine (GEM) has been the recommended first-line drug for patients with pancreatic ductal adenocarcinoma cancer (PDAC) for the last twenty years. However, GEM-based treatment has failed in many patients because of the drug resistance acquired during tumorigenesis and development. To override resistance to GEM in pancreatic cancer, we developed a visualisable, photothermally controlled, drug release nanosystem (VPNS). This nanosystem has NaLuF4:Nd@NaLuF4 nanoparticles as the luminescent core, octabutoxyphthalocyanine palladium(ii) (PdPc) as the photothermal agent, and phosphorylated gemcitabine (pGEM) as the chemodrug. pGEM, one of the active forms of GEM, can circumvent the insufficient activation of GEM in cancer cell metabolism. The NaLuF4:Nd@NaLuF4 nanoparticles were employed to visualise the tumor lesion in vivo by their near-infrared luminescence. The near-infrared light-triggered photothermal effect from PdPc could trigger the release of pGEM loaded in a thermally responsive ligand and simultaneously enable photothermal cancer treatment. This work presents an effective method that suppresses the growth of tumour cells with dual-mode treatment and enables the improved treatment of orthotopic nude mice afflicted with pancreatic cancer.

RGDV-modified gemcitabine: a nano-medicine capable of prolonging half-life, overcoming resistance and eliminating bone marrow toxicity of gemcitabine.

BACKGROUND: Gemcitabine has been widely used as a chemotherapeutic drug. However, drug resistance, short half-life and side effects seriously decrease its chemotherapeutic efficacy. PURPOSE: The object of preparing RGDV-gemcitabine was to prolong the half-life, to overcome drug resistance and to eliminate bone marrow toxicity of gemcitabine. METHODS: Arg-Gly-Asp-Val was coupled with gemcitabine, forming 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo-lan-2-yl]pyrimidin-2-one (RGDV-gemcitabine) involving 9-step reactions. The advantages of RGDV-gemcitabine to gemcitabine were demonstrated by a series of assays, such as in vitro half-life assay, in vitro drug resistance assay, in vivo anti-tumor assay, in vivo kidney toxicity assay, in vivo liver toxicity assay and in vivo marrow toxicity assay. The nano-features of RGDV-gemcitabine were visualized by TEM, SEM and AFM images. The tumor-targeting action and release of RGDV-gemcitabine were evidenced by FT-MS spectra. RESULTS: Half-life and anti-tumor activity of RGDV-gemcitabine were 17-fold longer and 10-fold higher than that of gemcitabine, respectively. RGDV-gemcitabine, but not gemcitabine, showed no kidney toxicity, no liver toxicity, no marrow toxicity and no drug resistance. The advantages attributed to the nanofeatures of RGDV-gemcitabine were targeting tumor tissue and releasing gemcitabine in tumor tissue. CONCLUSION: RGDV-gemcitabine successively overcame the defects of gemcitabine and provided a practical strategy of nano-medicine.

Polygemcitabine nanogels with accelerated drug activation for cancer therapy.

To overcome the slow activation of gemcitabine, we synthesized a DNA-like polygemcitabine (Ge10) strand through solid-phase synthesis, which not only undergoes rapid intracellular degradation to generate active gemcitabine derivatives, but can also self-assemble into nanogels through molecular recognition, rendering them as promising self-delivered nanodrugs for cancer therapy.

4-N-Alkanoyl and 4-N-alkyl gemcitabine analogues with NOTA chelators for 68-gallium labelling.

The conjugation of 4-N-(3-aminopropanyl)-2'-deoxy-2',2'-difluorocytidine with 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bn-NOTA) ligand in 0.1 M Na2CO3 buffer (pH 11) at ambient temperature provided 4-N-alkylgemcitabine-NOTA chelator. Incubation of latter with excess of gallium(III) chloride (GaCl3) (0.6 N AcONa/H2O, pH = 9.3) over 15 min gave gallium 4-N-alkylgemcitabine-NOTA complex which was characterized by HRMS. Analogous [68Ga]-complexation of 4-N-alkylgemcitabine-NOTA conjugate proceeded with high labeling efficiency (94%-96%) with the radioligand almost exclusively found in the aqueous layer (∼95%). The high polarity of the gallium 4-N-alkylgemctiabine-NOTA complex resulted in rapid renal clearance of the 68Ga-labelled radioligand in BALB/c mice.

Modification of the aminopyridine unit of 2'-deoxyaminopyridinyl-pseudocytidine allowing triplex formation at CG interruptions in homopurine sequences.

The antigene strategy based on site-specific recognition of duplex DNA by triplex DNA formation has been exploited in a wide range of biological activities. However, specific triplex formation is mostly restricted to homo-purine strands within the target duplex DNA, due to the destabilizing effect of CG and TA inversion sites where there is an absence of natural nucleotides that can recognize the CG and TA base pairs. Hence, the design of artificial nucleosides, which can selectively recognize these inversion sites with high affinity, should be of great significance. Recently, we determined that 2-amino-3-methylpyridinyl pseudo-dC (3MeAP-ΨdC) possessed significant affinity and selectivity toward a CG inversion site and showed effective inhibition of gene expression. We now describe the design and synthesis of new modified aminopyridine derivatives by focusing on small chemical modification of the aminopyridine unit to tune and enhance the selectivity and affinity toward CG inversion sites. Remarkably, we have newly found that 2-amino-4-methoxypyridinyl pseudo-dC (4OMeAP-ΨdC) could selectively recognize the CG base pair in all four adjacent base pairs and form a stable triplex structure against the promoter sequence of the human gene including multiple CG inversion sites.

Gemcitabine analogues with 4-N-alkyl chain modified with fluoromethyl ketone group.

Gemcitabine analogues with a lipophilic 4-N-alkyl chain bearing a terminal ß-keto sulfonate moiety suitable for fluorination compatible with 18F-radiolabeling have been explored. Displacement of p-toluenesulfonylamino in protected 4-N-tosylgemcitabine with 1-amino-10-undecene gave 4-N-(10-undecenyl)-3',5'-di-O-benzoyl-2'-deoxy-2',2'-difluorocytidine. Oxidation of the terminal double bond in the latter with OsO4/NMO afforded 4-N-(10,11-dihydroxyundecanyl) derivative. Regioselective sulfonation of primary hydroxyl followed by oxidation of secondary hydroxyl with Collin's reagent yielded desired ß-keto sulfonate analogues 8 or 9. Subsequent displacement of the mesylate or tosylate group with KF in the presence of Kryptofix 2.2.2. or 18-crown-6 ether followed by deprotection with NH3/MeOH gave 4-N-(11-fluoro-10-oxoundecanyl)-2'-deoxy-2',2'-difluorocytidine 11.

Nucleoside-Based Self-Assembling Drugs for Localized Drug Delivery.

We have synthesized a range of gelators based on the nucleoside analogues gemcitabine and lamivudine, characterizing representative gels from the series using rheology and transmission electron microscopy. Growth inhibition studies of gemcitabine derivatives confirmed the feasibility of these compounds as novel treatments, indicating the potential of nucleoside-based gelators for localized drug delivery.

Selectively Targeted Anti-Neoplastic Cytotoxicity of Three Immunopharmaceuticals with Covalently Bound Fludarabine, Gemcitabine and Dexamethasone Moieties Synthesized Utilizing Organic Chemistry Reactions in a Multi-Stage Regimen.

BACKGROUND: Unintentional passive diffusion of conventional small molecular weight pharmaceuticals across intact membranes of normal healthy cells in tissues and organ systems induces sequelae that limit therapeutic dosage and duration of administration. Selective "targeted" delivery of pharmaceuticals is a molecular strategy that can potentially provide heightened margins-of-safety with greater potency and improved efficacy. MATERIALS AND METHODS: Monophosphate analogs of fludarabine, gemcitabine, and dexamethasone were combined with a carbodiimide reagent in the presence of imidazole to produce reactive intermediates that were subsequently covalently bound to monoclonal anti-IGF-1R or anti-EGFR IgG-immunoglobulin. The resulting covalent immunopharmaceutical end-products, fludarabine-(5'-phosphoramidate)-[anti-IGF-1R], gemcitabine-(5'- phosphoramidate)-[anti-IGF-1R], and dexamethasone-(C21-phosphoramidate)-[anti-EGFR] were evaluated by SDS-PAGE/chemiluminescent autoradiography (fragmentation/polymerization detection), UV spectrophotometric absorbance (purity; molar-incorporation-index), cell-ELISA (retained selective binding-avidity), and cell vitality-viability (selectively "targeted" anti-neoplastic cytotoxicity). RESULTS: Maximum selectively "targeted" anti-neoplastic cytotoxicity of fludarabine-(5'-phosphoramidate)-[anti- IGF-1R], gemcitabine-(5'-phosphoramidate)-[anti-IGF-1R], and dexamethasone-(C21-phosphoramidate)-[anti- EGFR] was detected at the pharmaceutical-equivalent concentrations of 10-5 M (94.7%), 10-7 M (93.1%), and 10-7 M (64.9%) respectively. DISCUSSION: Organic chemistry reactions were optimized in a template multi-stage synthesis regimen for fludarabine-( 5'-phosphoramidate)-[anti-IGF-1R], gemcitabine-(5'-phosphoramidate)-[anti-IGF-1R], and dexamethasone-( C21-phosphoramidate)-[anti-EGFR]. Attributes of the synthesis regimen include; [-i-] covalent bonding of pharmaceutical moeities at high molar incorporation indexes, [-ii-] implementation of organic chemistry reactions in a non-dedicated synthesis regimen allowing component substitution and [-iii-] optional preservation of presynthesized amine-reactive pharmaceutical intermediates for on-demand immunopharmaceutical synthesis. Attributes of the covalent immunopharmaceuticals are; absence of any synthetically introduced chemical groups, retained IgG-immunoglobulin binding-avidity and potent selective "targeted" anti-neoplastic cytotoxic potency. Under in-vivo conditions, supplemental anti-neoplastic cytotoxicity is realized through trophic receptor inhibition and activation of multiple cytotoxic host immune responses.

Gemcitabine, Pyrrologemcitabine, and 2'-Fluoro-2'-Deoxycytidines: Synthesis, Physical Properties, and Impact of Sugar Fluorination on Silver Ion Mediated Base Pairing.

The stability of silver-mediated "dC-dC" base pairs relies not only on the structure of the nucleobase, but is also sensitive to structural modification of the sugar moiety. 2'-Fluorinated 2'-deoxycytidines with fluorine atoms in the arabino (up) and ribo (down) configuration as well as with geminal fluorine substitution (anticancer drug gemcitabine) and the novel fluorescent phenylpyrrolo-gemcitabine (ph PyrGem) have been synthesized. All the nucleosides display the recognition face of naturally occurring 2'-deoxycytidine. The nucleosides were converted into phosphoramidites and incorporated into 12-mer oligonucleotides by solid-phase synthesis. The addition of silver ions to DNA duplexes with a fluorine-modified "dC-dC" pair near the central position led to significant duplex stabilization. The increase in stability was higher for duplexes with fluorinated sugar residues than for those with an unchanged 2'-deoxyribose moiety. Similar observations were made for "dC-dT" pairs and to a minor extent for "dC-dA" pairs. The increase in silver ion mediated base-pair stability was reversed by annulation of a pyrrole ring to the cytosine moiety, as shown for 2'-fluorinated ph PyrGem in comparison with phenylpyrrolo-dC (ph PyrdC). This phenomenon results from stereoelectronic effects induced by fluoro substitution, which are transmitted from the sugar moiety to the silver ion mediated base pairs. The extent of the effect depends on the number of fluorine substituents, their configuration, and the structure of the nucleobase.

5-Formyl- and 5-Carboxydeoxycytidines Do Not Cause Accumulation of Harmful Repair Intermediates in Stem Cells.

5-Formyl-dC (fdC) and 5-carboxy-dC (cadC) are newly discovered bases in the mammalian genome that are supposed to be substrates for base excision repair (BER) in the framework of active demethylation. The bases are recognized by the monofunctional thymine DNA glycosylase (Tdg), which cleaves the glycosidic bond of the bases to give potentially harmful abasic sites (AP-sites). Because of the turnover of fdC and cadC during cell state transitions, it is an open question to what extent such harmful AP-sites may accumulate during these processes. Here, we report the development of a new reagent that in combination with mass spectrometry (MS) allows us to quantify the levels of AP-sites. This combination also allowed the quantification of ß-elimination (ßE) products, which are repair intermediates of bifunctional DNA glycosylases. In combination with feeding of isotopically labeled nucleosides, we were able to trace the intermediates back to their original nucleobases. We show that, while the steady-state levels of fdC and cadC are substantially increased in Tdg-deficient cells, those of both AP- and ßE-sites are unaltered. The levels of the detected BER intermediates are 1 and 2 orders of magnitude lower than those of cadC and fdC, respectively. Thus, neither the presence of fdC nor that of cadC in stem cells leads to the accumulation of harmful AP- and ßE-site intermediates.

Aminopyridinyl-Pseudodeoxycytidine Derivatives Selectively Stabilize Antiparallel Triplex DNA with Multiple CG Inversion Sites.

The sequence-specific formation of triplex DNA offers a potential basis for genome-targeting technologies. In an antiparallel triplex DNA, the sequence-specificity is established by the formation of specific base triplets (G-GC, A-AT, and T-AT) between a triplex-forming oligonucleotide (TFO) and a duplex DNA. However, there are no natural nucleosides that can selectively recognize the inverted CG and TA base pairs. Therefore, the recognition of the CG and TA inversion sites to form a stable triplex DNA has been a long-standing goal for the triplex-forming technology. We now describe the design and synthesis of pseudo-deoxycytidine (ΨdC) derivatives for selective recognition of the CG base pair to expand the triplex-forming sequence. The aminopyridine-bearing ΨdC derivatives showed high selectivity and affinity toward the CG base pair in all neighboring base contexts. Remarkably, 3-methyl-2-aminopyridinyl-ΨdC ((Me) AP-ΨdC) formed a stable triplex with the promoter sequence of the hTERT gene containing four CG inversion sites, and effectively inhibited its transcription in human cancer cells. Thus, (Me) AP-ΨdC is expected to serve as a new starting point of triplex-forming oligonucleotides for a wide variety of genome-targeting applications.