Design and synthesis of an environment-sensitive 3-methyleneisoindolin-1-one fluorophore for labeling DNA-interacting proteins.

We designed 6-dimethylamino 3-methyleneisoindolin-1-one as an environment-sensitive fluorophore, examining its applications for protein labeling. Synthesized 3-methyleneisoindolin-1-one exhibits solvatochromic fluorescence (; 472 nm in 2-PrOH, 512 nm in HO). A positive linear dependence between and solvent dielectric constant (DC), as well as between Stokes shift and DC, and a negative correlation between fluorescence quantum yield and DC are observed in protic solvents.

Structure-activity relationship study of nitrogen signaling factors.

We have synthesized 10 analogs of oxylipins, which are nitrogen signaling factors (NSFs) that mediate cell-to-cell communication in the fission yeast Schizosaccharomyces pombe, and evaluated their structure-activity relationships with the aim of developing molecular probes for NSFs. We found that the OH or OAc group at C10 could be replaced with a compact amide (17) or carbamate (19). Introducing an alkyne as a detection tag at C10 led to decreased, though still sufficient, activity.

Fluorescence Tumor-Imaging Using a Thermo-Responsive Molecule with an Emissive Aminoquinoline Derivative.

We synthesized (2,4-trifluoromethyl-7--bis(2,5,8,11-tetraoxatridecane-13-yl)-aminoquinoline) TFMAQ-diEg4, an emissive aminoquinoline derivative that incorporated two tetraethyleneglycol chains into an amino group. TFMAQ-diEg4 showed fluorescence and thermo-responsive properties accompanied by a lower critical solution temperature (LCST), due to the introduction of the oligoethylene glycol chain. This thermo-responsive LCST behavior occurred at the border of a cloud point.

7-Hydroxy-3-methyleneisoindolin-1-one as a New ESIPT-Fluorescent Probe to Monitor Aqueous Environments.

A 7-hydroxy derivative of 3-methyleneisoindolin-1-one 1 was synthesized and its properties as a new fluorophore undergoing excited-state intramolecular proton transfer (ESIPT) were investigated. In alcohols and dimethylsulfoxide, 1 exhibited dual emission at ca. 380 and 525-540 nm when excited at ca.

Synthesis and Electron Paramagnetic Resonance Studies of Oligodeoxynucleotides Containing 2-N-tert-Butylaminoxyl-2'-deoxyadenosines.

Oligodeoxynucleotides (ODNs) containing 2-N-tert-butylaminoxyl-2'-deoxyadenosine (A*) residues were synthesized to allow accurate monitoring of adenine motion by EPR spectroscopy through the agency of direct linkage of the acyclic aminoxyl group to the nucleobase, and EPR studies of the ODNs in single- and double-stranded forms were performed. Upon duplex formation, peak broadening and decreases in peak height were observed in EPR spectra, and the synthesized ODNs were shown to be excellent monitors of hybridization. Comparison of peak height and the h /h signal ratio provided information on the relative mobility of A* in duplexes with different stability.

Site-Specific Turn-On Fluorescent Labeling of DNA-Interacting Protein Using Oligodeoxynucleotides That Modify Lysines To Produce 5,6-Dimethoxy 3-Methyleneisoindolin-1-one.

We have developed oligodeoxynucleotides (ODNs) that modify primary amines to produce 5,6-dimethoxy 3-methyleneisoindolin-1-one. Compared to the oxygen isosteric fluorophore, 4,5-dimethoxyphthalimide, this methyleneisoindolinone was more stable and exhibited an 85 nm blue-shifted fluorescent emission (λmax at 425 nm) with an intensity comparable to that of the phthalimide. Reaction of the DNA-binding domain of Escherichia coli DnaA protein with an ODN containing its binding sequence efficiently afforded a modified fluorescent protein at a specific lysine residue in the proximity of the ODN.

Bioconjugation of Oligodeoxynucleotides Carrying 1,4-Dicarbonyl Groups via Reductive Amination with Lysine Residues.

We evaluated the efficacy of bioconjugation of oligodeoxynucleotides (ODNs) containing 1,4-dicarbonyl groups, a C4'-oxidized abasic site (OAS), and a newly designed 2'-methoxy analogue, via reductive amination with lysine residues. Dicarbonyls, aldehyde and ketone at C1- and C4-positions of deoxyribose in the ring-opened form of OAS allowed efficient reaction with amines. Kinetic studies indicated that reductive amination of OAS-containing ODNs with a proximal amine on the complementary strand proceeded 10 times faster than the corresponding reaction of an ODN containing an abasic site with C1-aldehyde.

Production and characterization of highly specific monoclonal antibodies to D-glutamic acid.

Most of the functions of D-amino acids (D-AA) remain unclear because of little analytic methods for specific detection/determination. In this study, a highly specific monoclonal antibody to D-glutamic acid (D-Glu-MAb) was produced using a hybridoma method. Characterization of D-Glu-MAb by indirect enzyme-linked immunosorbent assay (ELISA) revealed that it has high selectivity against D-Glu-glutaraldehyde (GA) conjugates, while no cross-reaction was observed when 38 other kinds of AA-GA conjugates were used.

Difluoro-C4'-oxidized abasic site for efficient amine modification in biological systems.

An oligodeoxynucleotide (ODN) containing a 2',2'-difluorinated analogue of a C4'-oxidized abasic site (C4'-OAS) was designed for the amine modification of biomolecules that interact with nucleic acids. In contrast to the parent C4'-OAS, which yielded amine-modified products accompanied by DNA strand scission, the ODN containing the difluoro C4'-OAS efficiently yielded products carrying ODNs. The amine modification proceeded without additional reagents being required and might be applicable to reactions in biological systems.

Dialkyl bisphosphonate platinum(II) complex as a potential drug for metastatic bone tumor.

Bisphosphonates have high affinity for hydroxyapatite (HA), which is abundantly present in bone. Also, platinum complexes are known that have a wide spectrum of antitumor activities. The conjugate of bisphosphonate and a platinum complex might have HA affinity and antitumor activity, and become a drug for metastatic bone tumor.

Photochemical generation and reaction of 2'-substituted analogues of C4'-oxidized abasic lesion.

The C4'-oxidized abasic site (1) is one of the oxidatively damaged DNA lesions induced by antitumor bleomycins. The reactivity of 1 with amine under neutral conditions giving lactam 2 and its structural similarity to unmodified DNA suggested the possibility that ODN containing 1 could react with proteins which interact with DNA at lysine residue. In order to provide nucleic acid analogues with useful lysine modifying reactivity, we planned to study reaction of 2'-substituted analogues of 1, such as 3-5, with amine and their corresponding caged precursors 6-8 were synthesized.

Evaluation of mobility of 2-N-tert-butylaminoxyladenosine incorporated into oligodeoxynucleotides.

We synthesized oligodeoxynucleotide (ODN, 3) containing 2-N-tert-butylaminoxyladenosine (1) and studied EPR spectra of 3 and its duplexes. The h(+)/h(0) values in the EPR spectra of duplexes between 3 and 5-8 well correlated with Tm values. We also synthesized ODN 4 containing 2, which has a cyclic aminoxyl via a linker, to compare with ODN 3.

Water-proton relaxivities of DNA oligomers carrying TEMPO radicals.

5-Uridine derivative carrying a TEMPO radical (UST) was prepared and its single strand (ssUST) and a double strand (dsUST) with its complementary strand were obtained. Similarly, single strands carrying two and five radicals (ssUST2 and ssUST5, respectively) and the corresponding double strands (dsUST2 and dsUST5) were prepared. Their electron paramagnetic resonance (EPR) spectra showed typical anisotropic broadening in the high field line.

Dynamics of 2-N-tert-butylaminoxyladenosine incorporated into oligodeoxynucleotides.

We synthesized oligodeoxynucleotide (ODN, 3) containing 2-N-tert-butylaminoxyladenosine (1) and studied EPR spectra of 1 and its duplexes with varied sequences. Duplex formation resulted in a broadening of spectrum and a significant decrease in peak-to-peak height and peak height ratio values. The h(+)/ho values in EPR spectra well correlated with stability of duplex which indicated ODN containing 1 has the potential to monitor DNA structure.

Synthesis of nitroxyl radicals for Overhauser-enhanced magnetic resonance imaging.

Non-invasive measurement and visualization of free radicals in vivo would be important to clarify their roles in the pathogenesis of free radical-associated diseases. Nitroxyl radicals can react with free radicals and be derivatized to achieve specific cellular/subcellular localizing capabilities while retaining the simple spectral features useful in imaging. Overhauser-enhanced magnetic resonance imaging (OMRI), which is a double resonance technique, creates images of free radical distributions in small animals by enhancing the water proton signal intensity via the Overhauser Effect.

Photochemical generation of oligodeoxynucleotide containing a C4'-oxidized abasic site and its efficient amine modification: dependence on structure and microenvironment.

Bleomycin-induced oxidative DNA damage under limited oxygen conditions results in the formation of the C4'-oxidized abasic site (1). We synthesized the oligodeoxynucleotides (ODN) 5, which contains 4'-o-nitrobenzyloxythymidine (3), and 6, which contains 2-nitrobenzyloxy-4'-methoxy-2'-deoxy-d-ribofuranoside (4), as the caged precursors of 7, an ODN containing 1, to study its reactivity with amines. Photoirradiation of the single- and double-stranded 5 led to the formation of 7.

Studies on DNA dynamics using 2-N-tert-butylaminoxylpurines.

2-N-tert-Butylaminoxylpurines (1) and (2) were synthesized from 2'-deoxy-6-chloropurine derivative by lithiation strategy. Effects of motion of 1 and 2 on EPR spectra were studied by EPR measurement in sucrose solution at various temperatures. The single stranded and duplexed 15-mers containing 1 showed the clear difference in the EPR spectra to indicate 1 has the potential to accurately study the dynamics of purine residues.

The photoreactivity of the caged precursors of oligonucleotides containing C4'-oxidized abasic site and reaction of the generated lesion.

The C4'-oxidized abasic site (1) is one of the oxidatively damaged DNA lesions by antitumor bleomycins (BLMs) and the acyclic keto aldehyde incorporated in DNA might react with DNA interacting biomolecules. We synthesized oligodeoxynuceotides 3-caged-1 and 3-caged-2 as caged precursors of 3, an ODN containing 1, to study the reactivities of 3 with amine. Irradiation of double stranded 3-caged-1 led to efficient formation of 3 compared to its single stranded form.

Photochemical generation of C4'-oxidized abasic site containing oligonucleotide and its amine modification reaction.

We studied photochemical generation of oligodeoxynucleotide (ODN) containing C4'-oxidized abasic site (1) from its caged precursor 3. Photoirradiation of 3 at 365 nm followed by amine treatment under neutral conditions afforded the lactam (2) efficiently. Duplexed ODN 3 was converted to 1 faster and more efficiently than single stranded 3, whereas amine treatment of 1 formed from single stranded 3 resulted in slightly faster lactam formation than with duplex.

Amine modification reaction of C4'-oxidized abasic site generated from its caged precursors.

A method for the generation of ODNs containing a C4'-oxidized abasic site (C4'-OAS) from "photocaged" ODNs was investigated. We synthesized C4'-caged ODN 1 and C1'-caged ODN 2 bearing an o-nitrobenzyloxy group at the C-4' position and the C-1' position, respectively. The duplex stabilities of 1 and 2, with their complementary ODNs were studied by Tm measurements.

Properties and EPR studies of oligodeoxynucleotides containing 2-N-tert-butylaminoxylpurines.

Oligodeoxynucleotide (ODN; 1) containing 2-N-tert-butylaminoxyladenosine (2) was synthesized by the conventional phosphoramidite method. The thermal stability and structure of the duplex of ODN 1 and the complementary strand were elucidated by UV-melting temperature measurements and CD experiments. In phosphate buffer, ODN 1 showed a three line EPR spectrum.

Synthesis of oligonucleotide containing 4'-o-nitrobenzyloxythymidine and its reactivity.

We synthesized oligonucleotide (1) containing 4'-o-nitrobenzyloxythymidine (2), which was prepared by reaction of 4',5'-unsaturated thymidine with m-CPBA followed by Lewis-acid catalyzed addition of o-nitrobenzylalcohol. The duplex stability of 1 with the complementary oligonucleotide was studied by Tm measurement. Photochemical reaction of 1 to generate C4'-oxidized abasic site containing oligonucleotide (3) was studied.

Molecular design of novel spin-labeled nucleosides directly attached base moiety.

N-tert-Butylaminoxyl nucleosides (1 and 2) were synthesized starting from 6-chloropurine derivative (3). These compounds are expected as a new tool to elucidate structure of DNA. Their properties and pH-dependent change of EPR spectra were studied.

Photochemical generation of C4'-oxidized abasic site containing oligodeoxynucleotide and its efficient amine modification.

[Structure: see text] We synthesized oligodeoxynucleotide (ODN, 3), which contains 4'-o-nitrobenzyloxythymidine (4) as a caged precursor of C4'-oxidized abasic site (1). Photoirradiation of 3 at 365 nm followed by amine treatment under neutral conditions afforded the lactam (2) efficiently. Duplexed ODN 3 was converted to 1 faster and more efficiently than single stranded 3, whereas amine treatment of 1 formed from single stranded 3 resulted in slightly faster lactam formation than with the duplex.