A Chimeric Nucleobase - Phenylazo Derivative as an Intrinsic Nucleobase Quencher

Molecular beacons are important bioanalytical probes which are most often constructed from a single-stranded oligonucleotide which has been labeled at opposite termini with a fluorophore and a quencher. When the fluorophore and quencher are in close proximity, no fluorescence is observed due to FRET (Fluorescence Resonance Energy Transfer). DABCYL (4-dimethylaminoazobenzene- 4\u27-carboxylic acid) has been used as a quencher in the molecular beacon to absorbs excitation energy from a fluorophore and to dissipate the energy as heat. However, DABCYL is unable to form a base-pair and is conventionally placed as an overhanging residue. This produces a derivative wherein the chromophore has substantial mobility and limits the types of other conjugates that can be prepared. In order to overcome these limitations, we have embarked on the synthesis of deoxyribonucleoside and peptide nucleic acid (PNA) analogue possessing DMPAU (5-[(4-dimethylaminophenyl) diazenyl]uracil) as the nucleobase. DMPAU has DABCYL-like properties due to the installation of an azo moiety at the 5-position of the uracil base. This base is designed to have the ability to form a complementary base pair with adenosine by canonical hydrogen bonding and also to quench the fluorescence emission in a molecular beacon construct. Both DMPAUridine and DMPAU PNA analogue are determined to have same UV-Vis absorbance ranges as DABCYL and reasonable quenching effect to the fluorophore

Detection of Active Caspase-3 in Mouse Models of Stroke and Alzheimer\u27s Disease with a Novel Dual Positron Emission Tomography/Fluorescent Tracer [68Ga]Ga-TC3-OGDOTA.

Apoptosis is a feature of stroke and Alzheimer\u27s disease (AD), yet there is no accepted method to detect or follow apoptosis in the brain in vivo. We developed a bifunctional tracer [Ga-68]Ga-TC3-OGDOTA containing a cell-penetrating peptide separated from fluorescent Oregon Green and Ga-68-bound labels by the caspase-3 recognition peptide DEVD. We hypothesized that this design would allow [Ga-68]Ga-TC3-OGDOTA to accumulate in apoptotic cells. In vitro, Ga-TC3-OGDOTA labeled apoptotic neurons following exposure to camptothecin, oxygen-glucose deprivation, and -amyloid oligomers. In vivo, PET showed accumulation of [Ga-68]Ga-TC3-OGDOTA in the brain of mouse models of stroke or AD. Optical clearing revealed colocalization of [Ga-68]Ga-TC3-OGDOTA and cleaved caspase-3 in brain cells. In stroke, [Ga-68]Ga-TC3-OGDOTA accumulated in neurons in the penumbra area, whereas in AD mice [Ga-68]Ga-TC3-OGDOTA was found in single cells in the forebrain and diffusely around amyloid plaques. In summary, this bifunctional tracer is selectively associated with apoptotic cells in vitro and in vivo in brain disease models and represents a novel tool for apoptosis detection that can be used in neurodegenerative diseases

Detection of active caspase-3 in mouse models of stroke and Alzheimer\u27s disease with a novel dual positron emission tomography/fluorescent tracer [ \u3csup\u3e68\u3c/sup\u3e Ga]Ga-TC3-OGDOTA

© 2019 Valeriy G. Ostapchenko et al. Apoptosis is a feature of stroke and Alzheimer\u27s disease (AD), yet there is no accepted method to detect or follow apoptosis in the brain in vivo. We developed a bifunctional tracer [ 68 Ga]Ga-TC3-OGDOTA containing a cell-penetrating peptide separated from fluorescent Oregon Green and 68 Ga-bound labels by the caspase-3 recognition peptide DEVD. We hypothesized that this design would allow [ 68 Ga]Ga-TC3-OGDOTA to accumulate in apoptotic cells. In vitro, Ga-TC3-OGDOTA labeled apoptotic neurons following exposure to camptothecin, oxygen-glucose deprivation, and β-amyloid oligomers. In vivo, PET showed accumulation of [ 68 Ga]Ga-TC3-OGDOTA in the brain of mouse models of stroke or AD. Optical clearing revealed colocalization of [ 68 Ga]Ga-TC3-OGDOTA and cleaved caspase-3 in brain cells. In stroke, [ 68 Ga]Ga-TC3-OGDOTA accumulated in neurons in the penumbra area, whereas in AD mice [ 68 Ga]Ga-TC3-OGDOTA was found in single cells in the forebrain and diffusely around amyloid plaques. In summary, this bifunctional tracer is selectively associated with apoptotic cells in vitro and in vivo in brain disease models and represents a novel tool for apoptosis detection that can be used in neurodegenerative diseases

Influence of C-5 substituted cytosine and related nucleoside analogs on the formation of benzo[a]pyrene diol epoxide-dG adducts at CG base pairs of DNA

Endogenous 5-methylcytosine (MeC) residues are found at all CG dinucleotides of the p53 tumor suppressor gene, including the mutational ‘hotspots' for smoking induced lung cancer. MeC enhances the reactivity of its base paired guanine towards carcinogenic diolepoxide metabolites of polycyclic aromatic hydrocarbons (PAH) present in cigarette smoke. In the present study, the structural basis for these effects was investigated using a series of unnatural nucleoside analogs and a representative PAH diolepoxide, benzo[a]pyrene diolepoxide (BPDE). Synthetic DNA duplexes derived from a frequently mutated region of the p53 gene (5′-CCCGGCACCC GC[15N3,13C1-G]TCCGCG-3′, + strand) were prepared containing [15N3, 13C1]-guanine opposite unsubstituted cytosine, MeC, abasic site, or unnatural nucleobase analogs. Following BPDE treatment and hydrolysis of the modified DNA to 2′-deoxynucleosides, N2-BPDE-dG adducts formed at the [15N3, 13C1]-labeled guanine and elsewhere in the sequence were quantified by mass spectrometry. We found that C-5 alkylcytosines and related structural analogs specifically enhance the reactivity of the base paired guanine towards BPDE and modify the diastereomeric composition of N2-BPDE-dG adducts. Fluorescence and molecular docking studies revealed that 5-alkylcytosines and unnatural nucleobase analogs with extended aromatic systems facilitate the formation of intercalative BPDE-DNA complexes, placing BPDE in a favorable orientation for nucleophilic attack by the N2 position of guanin

Synthesis and Photophysical Evaluation of New Fluorescent 7-Arylethynyl-7-Deazaadenosine Analogs

Three new fluorescent 7-deaza-2´-deoxyadenosine analogs were synthesized via the Sonogashira cross-coupling reaction of 7-iodo-7-deaza-2´-deoxyadenosine with 1-ethynylpyrene, 2-ethynyl-6-methoxynaphthalene, and 9-ethynylphenanthrene. The spectral properties of these analogs were evaluated in dioxane, EtOH, and H2O to determine their potential for use as environmentally sensitive fluorescent probes. All three analogs displayed large solvatofluorochromicity in H2O, relative to their emission wavelengths in dioxane or EtOH. Moreover, all three analogs exhibited microenvironmental sensitivity of their fluorescence emission intensity, being moderate to high quantum yields in dioxane and EtOH and significantly lower in H2O. Various attempts to perform domino cross coupling/annuation reactions on 7-deaza-7-alkynyladenine derivatives to form a new fused tricyclic adenine analog were unsuccessful.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Dysprosium(III) and thulium(III) complexes of DO3A-monoanilides: an investigation of electronic effects on their relaxometric and amide-based PARACEST properties

A series of 1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate monoamide (DO3A-monoanilide) complexes Dy3+ and Tm3+ were prepared and their magnetic properties evaluated in the context of their potential use as pH sensors. The ligands varied by para-substitution of the aniline moiety and represent electron-withdrawing and electron-donating groups. Only the Tm3+ complexes produced chemical exchange saturation transfer (CEST) spectra with CEST intensities due to the amide proton ranging from 1% to 8%. A maximum CEST signal was observed under slightly alkaline conditions (pH ∼8) when electron-donating groups were present, whereas the strongly electron-withdrawing nitro group produced a maximum CEST at neutral pH (pH = 7). The T1 and T2 relaxivities of the Dy3+ and Tm3+ complexes were also assessed. The T1 relaxivities of the Dy3+ and Tm3+ complexes were both low (r1 ≤ 0.3 mM−1 s−1, 25 °C, pH = 7) but, as expected, the Dy3+ complexes had much higher T2 relaxivities (r2 = 2–7 mM−1 s−1, 25 °C, pH = 7) as compared to the Tm3+-based chelates (r2 ≤ 0.09 mM−1 s−1, 25 °C, pH = 7)

Preliminary evaluation of PARACEST MRI agents for the detection of nitric oxide synthase

Several paramagnetic chemical exchange saturation transfer magnetic resonance imaging (PARACEST MRI) agents for the potential detection of nitric oxide synthase (NOS) have been synthesized and evaluated. These agents are based on an amino acid-or dipeptide-decorated DOTAM (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid amide) chelator possessing either Tm3+ or Dy3+. The amino acid and dipeptide decorated DOTAMs were designed such that the terminal amino acid pendant group was l-arginine, which may be converted to l-citrulline by NOS. Preliminary evaluation has revealed that some of the l-arginine-decorated complexes are recognized and metabolized by the NOS. Differences in the CEST properties between Dy3+-metallated l-arginine-and l-citrulline-modified complexes suggest that these might be suitable for imaging of the NOS enzymatic activity

Introduction of Peripheral Carboxylates to Decrease the Charge on Tm\u3csup\u3e3+\u3c/sup\u3e DOTAM-Alkyl Complexes: Implications for Detection Sensitivity and in Vivo Toxicity of PARACEST MRI Contrast Agents

A series of structurally modified Tm3+ DOTAM-alkyl complexes as potential PARACEST MRI contrast agents has been synthesized with the aim to decrease the overall positive charge associated with these molecules and increase their biocompatibility. Two types of structural modification have been performed, an introduction of terminal carboxylate arms to the alkyl side chains and a conjugation of one of the alkyl side chains with aspartic acid. Detailed evaluation of the magnetic resonance imaging chemical exchange contrast associated with the structurally modified contrast agents has been performed. In contrast to the acutely toxic Tm3+ DOTAM-alkyl complexes, the structurally modified compounds were found to be tolerated well during in vivo MRI studies in mice; however, only the aspartic acid modified chelates produced an amide proton-based PARACEST signal. (Figure Presented)

Influence of C-5 substituted cytosine and related nucleoside analogs on the formation of benzo[a]pyrene diol epoxide-dG adducts at CG base pairs of DNA

Endogenous 5-methylcytosine (MeC) residues are found at all CG dinucleotides of the p53 tumor suppressor gene, including the mutational ‘hotspots’ for smoking induced lung cancer. MeC enhances the reactivity of its base paired guanine towards carcinogenic diolepoxide metabolites of polycyclic aromatic hydrocarbons (PAH) present in cigarette smoke. In the present study, the structural basis for these effects was investigated using a series of unnatural nucleoside analogs and a representative PAH diolepoxide, benzo[a]pyrene diolepoxide (BPDE). Synthetic DNA duplexes derived from a frequently mutated region of the p53 gene (5′-CCCGGCACCC GC[15N3,13C1-G]TCCGCG-3′, + strand) were prepared containing [15N3, 13C1]-guanine opposite unsubstituted cytosine, MeC, abasic site, or unnatural nucleobase analogs. Following BPDE treatment and hydrolysis of the modified DNA to 2′-deoxynucleosides, N2-BPDE-dG adducts formed at the [15N3, 13C1]-labeled guanine and elsewhere in the sequence were quantified by mass spectrometry. We found that C-5 alkylcytosines and related structural analogs specifically enhance the reactivity of the base paired guanine towards BPDE and modify the diastereomeric composition of N2-BPDE-dG adducts. Fluorescence and molecular docking studies revealed that 5-alkylcytosines and unnatural nucleobase analogs with extended aromatic systems facilitate the formation of intercalative BPDE–DNA complexes, placing BPDE in a favorable orientation for nucleophilic attack by the N2 position of guanine.ISSN:1362-4962ISSN:0301-561