•Various environmentally sensitive fluorescent (ESF) nucleosides were synthesized.•These are first ESF nucleosides possessing a C2-substituted azadeazaadenine skeleton.•Compound 1c exhibited a remarkably high solvatochromicity (Δλ = 111 nm).•1c-containing DNA probes clearly discriminated an AP site in target DNA.We synthesized various C2-naphthylethynylated 8-aza-7-deaza-2′-deoxyadenosines 1a–1c as novel environmentally sensitive fluorescent (ESF) purine nucleosides. In particular, the N,N-dimethylamino-substituted derivative 1c exhibits a remarkably high solvatochromicity (Δλλ = 111 nm). 1c-containing oligodeoxynucleotide (ODN) probes clearly discriminated an apurinic/apyrimidinic site (AP site) in the complementary strand via a change in emission wavelength and intensity when hybridized with a target ODN.

A new environmentally responsive fluorescent nucleoside, 3-(naphthalen-1-ylethynyl)-3-deaza-2′-deoxyguanosine (3nzG), has been synthesized. The nucleoside, 3nzG, exhibited solvatochromic properties and when introduced into ODN probes it was able to recognize 2′-deoxycytidine in target strands by a distinct change in its emission wavelength through probing microenvironmental changes in the DNA minor groove. Thus, 3nzG has the potential for use as a fluorescent probe molecule for micro-structural studies of nucleic acids including the detection of single-base alterations in target DNA sequences.

A novel fluorescent benzo[g]imidazo[4,5-c]quinoline nucleoside BIQA (1) comprising a 3-deaza-2′-deoxyadenosine skeleton was developed and used to monitor BIQA–C base-pair formation in oligodeoxynucleotide (ODN) duplexes. The newly synthesized BIQA exhibited distinct photophysical properties associated with its protonated/deprotonated forms (monomer: pKa 6.2) via dramatic changes in its absorption and fluorescence spectra. In ODN duplexes, the induced protonation of BIQA occurred, even under alkalescent conditions when cytosine was the opposite base on the complementary strand; the resulting BIQA–C base pairs were stable. By monitoring the protonation of BIQA under neutral and alkalescent conditions, we could clearly discriminate cytosine through spectral changes in absorption and fluorescence. Similarly, we found that the demonstrated 3-deaza-2′-deoxyadenosine 3zA forms a stable base pair with cytosine via N1 protonation in ODN duplexes under neutral and acidic conditions (pH < 7.0). At lower pH values, 3zA-containing ODNs could clearly discriminate cytosine through melting temperature (Tm) measurements. Therefore, ODN probes containing indicator nucleosides, such as BIQA and 3zA, exhibit great potential as bioprobes for genetic analysis and structural studies of nucleic acids.

8-Aza-3,7-dideaza-2′-deoxyadenosine 1 and its C3-naphthylethynylated derivative 3n7zA (2) comprising a 8-aza-3,7-dideazapurine (pyrazolo[4,3-c]pyridine) skeleton were synthesized for the first time. In particular, nucleoside 3n7zA (2) exhibited environmentally sensitive intramolecular charge transfer (ICT) emission because of electron transition in the coplanar conformer formed by nucleobase and naphthalene moieties. Its incorporation into oligodeoxynucleotide (ODN) probes enable a clear identification of a perfectly matched thymine (T) in the complementary strand by a distinct change in the emission wavelength. In addition, the fluorescence emission of the duplexes containing a cytosine/guanine (C/G) base pair flanking 3n7zA (2) was strongly quenched by guanine only when the opposite base of the modified nucleoside was mismatched, enhancing its base identification ability. Thus, ODN probes containing 3n7zA (2) acted as effective reporter probes for homogeneous single nucleotide polymorphism (SNP) typing.

Pyrene-labeled 3-deaza-2′-deoxyadenosine comprising a non-π-conjugated linker py3zA (1) was synthesized and its photophysical properties were investigated. Oligodeoxynucleotide (ODN) probes containing py3zA (1) exhibited remarkable fluorescence quenching only when the opposite base of the complementary strand was the perfectly matched thymine. Such fluorescence quenching-based ODN probes exhibited excellent on-off switching properties, making them useful tools for single nucleotide polymorphism (SNP) genotyping and for the identification of target genes and structural studies of nucleic acids.

A novel environmentally sensitive fluorescent (ESF) purine nucleoside, cnaA, was synthesized and its photophysical properties were investigated. The base-modified fluorescent nucleoside cnaA exhibited remarkable solvatochromicity and environmentally sensitive dual fluorescence. By using cnaA, we have developed highly thymine (T) selective fluorescent DNA probes that can sense the corresponding T in a target DNA/RNA sequence, as measured by a distinct change in emission wavelength.

C7-naphthylethynylated 8-aza-7-deaza-2′-deoxyguanosine naG was synthesized and its photophysical properties were examined. The fluorescent nucleoside exhibited solvatofluorochromic properties (Δλflmax = 67 nm). An ODN probe containing naG forms a stable base pair only with C and discriminates structural changes such as mismatches and deletions by a distinct change in its emission wavelength.

We synthesized environmentally sensitive fluorescent (ESF) 7-deaza-2′-deoxyadenosine derivatives including ethynylanthracene substituted atzA (1) and ethynylnaphthalene substituted nzA (2a), cnzA (2b), anzA (2c), and dnzA (2d), and investigated their photophysical properties. Among them, only push–pull type cyano- and acetyl-substituted naphthylethynylated 7-deaza-2′-deoxyadenosine, cnzA (2b) and anzA (2c), exhibited remarkable solvatofluorochromic properties (Δλ = 71 and 63 nm, respectively). We incorporated non-solvatofluorochromic atzA (1) and solvatofluorochromic cnzA (2b) into oligodeoxynucleotides and found that cnzA (2b) forms a stable base pair with both thymine and cytosine, being accompanied by a change of fluorescence intensity. Such ESF nucleosides can be used for studying structures and functions of nucleic acids.

We synthesized various C8-naphthylethynylated 2′-deoxyadenosine derivatives and investigated their photophysical properties. Among them, cyano- and N,N-dimethylamino-substituted 8-naphthylethynylated 2′-deoxyadenosine derivatives (cnA and dnA) showed strong fluorescence with high quantum yields and a remarkable solvatofuorochromicity. In particular, fluorescence of N,N-dimethylamino-substituted 2,6dnA was not quenched by neighboring guanines (Gs) when incorporated in DNA duplexes, in contrast to cnA. We developed a new fluorescent probe containing 2,6dnA that can be used for the detection of target DNA via a bulge formation regardless of the neighboring sequences.

5-(1-Naphthalenylethynyl)-2′-deoxyuridine (NU) and 5-[(4-cyano-1-naphthalenyl)ethynyl]-2′-deoxyuridine (CNU) were synthesized and incorporated into oligodeoxynucleotides. Fluorescence emissions of modified duplexes containing double NU were efficiently quenched depending upon the sequence pattern of the naphthalenes in DNA major groove, as compared to the duplex possessing single NU. When one of the naphthalene moieties has a cyano substituent, the exciplex emission from the chromophores in DNA major groove was observed at longer wavelength.

We demonstrated an intriguing method to discriminate adenine by incident appearance of an intense new emission via exciplex formation in hybridization of target DNA with newly designed fluorescent 8-arylethynylated deoxyguanosine derivatives. We described the synthesis of such highly electron donating fluorescent guanosine derivatives and their incorporation into DNA oligomers which may be used for the structural study and the fluorometric analysis of nucleic acids.

We synthesized various pH-responsive fluorescent deoxyuridine derivatives (1a–g). These fluorescent nucleosides exhibited distinctive fluorescence at 470–600 nm in aqueous solvents containing methanol only at acidic to neutral pH values. In particular, 1f exhibited strong fluorescence only at pH range of 3.1–7.2 with a pKa of 6.1. Such pH-sensitive fluorescent nucleosides can be used as ‘on–off’ fluorescence switch for monitoring pH change in biological systems, particularly for cancer cell detection.Structures of fluorescent pH responsive nucleosides.

We synthesized various substituted 8-styryl-2′-deoxyguanosine and 8-styryl-2′-deoxyadenosine derivatives. Among them only acetyl substituted 8-styryl-2′-deoxyguanosine analog 5b showed a remarkable solvatochromicity (Δλmaxem=91nm),that is, strong fluorescence at 477 nm in 1,4-dioxane, but in methanol the fluorescence was red shifted to 558 nm with very low intensity. Such environmentally sensitive solvatochromic fluorescent guanosine analogs may be useful as a sensor for investigating interactions of DNA with DNA binding proteins.Chemical structures of aromatic fluorophore substituted purine nucleosides.

The synthesis and photophysical property of novel solvatochromic pyrene derivative, Apa5 and fluorescent guanine ApaG8 were described. These newly synthesized fluorescent pyrene derivatives exhibited solvent polarity dependent fluorescence at longer wavelengths. Such environmentally sensitive pyrene derivatives can be used as a reporter probe that is sensitive to the changes in the microenvironment around DNA either in vitro or in vivo.

We have synthesized two novel push–pull-type fluorescent 7-deazapurine nucleosides, CNZA and CNZG, and investigated their photophysical properties. In particular, CNZA was found to exhibit a remarkable solvatofluorochromicity (Δλfl.max = 60 nm). We incorporated CNZA into oligonucleotides and found that CNZA can form a stable base pair with both thymine and cytosine. Such environmentally sensitive fluorescent nucleosides have a potential as a fluorescence sensor for structural studies of nucleic acids.

We have developed novel push–pull-type 8-arylbutadienyl 2′-deoxyguanosine derivatives, ABG (1a) and CBG (1b). These nucleosides exhibit strong solvent polarity dependent fluorescence emission at long wavelength (ca. 490–550 nm). These environmentally sensitive fluorescent deoxyguanosines are powerful tools for structural studies of nucleic acids and also in molecular diagnostics.

2-(1-Naphthalenylethynyl)-2′-deoxyadenosine (NA) was synthesized and incorporated into oligodeoxynucleotides. DNA duplexes containing newly designed 5′-NAT-3′/3′-TNA-5′ base pairs are considerably stabilized than unmodified duplexes by stacking interaction of naphthalene rings in the narrow minor groove as characterized by a new emission at longer wavelength and exciton coupled CD signals.

We synthesized novel push–pull-type fluorescent guanosine derivatives, CNG and AcG containing 1,6- and 2,7-disubstituted pyrene chromophores. 1,6-Disubstituted pyrene derivatives, 1,6-CNG (3b) and 1,6-AcG (3c), exhibited highly solvatochromic fluorescence emission at longer wavelength (∼540 nm). The environmentally sensitive fluorescent deoxyguanosines such as 3b and 3c can be used as powerful tools for structural studies of nucleic acids and molecular diagnostics.

We have synthesized various substituted 8-arylethynylated 2′-deoxyguanosine derivatives. Among them, acetyl substituted deoxyguanosine analogue 4c showed a remarkable solvent dependent fluorescence property, that is, an intense fluorescence in non-polar solvents but extremely weak fluorescence in polar solvents like methanol. By using solvatofluorochromic deoxyguanosine analogue 4c, we have developed highly thymine (T) selective fluorescent DNA probes that can sense T opposite 4c in a target DNA regardless of the flanking sequences. We were able to demonstrate that 4c can be used as a T specific base-discriminating fluorescent (BDF) nucleoside in homogeneous fluorescence assay.

2- and 9-Anthracenecarboxamide labeled 2′-deoxyuridines were synthesized and their photophysical properties were examined. These oligonucleonucleotide probes are capable of detecting adenine base on a target DNA sequence. It was also found that 2-anthracene based oligonucleotide probe is more efficient than the corresponding 9-anthracene based oligonucleotide in the application for DNA chip based SNP detection, due to its longer emission wavelength and high fluorescence intensity.

A novel FRET based strategy for DNA sequence analysis utilising base-discriminating fluorescence (BDF) nucleoside, PyU/2-AntU, as donor in the dual-labelled oligonucleotide probe is reported; a selective/specific emission from acceptor, was observed upon excitation at the donor, only when the opposite base of the “smart” fluorescently labeled BDF nucleoside, PyU/2-AntU, is adenine on the complementary target sequence.

G-quenched MBs are devised from readily available starting materials and used for sequence specific DNA detection with high efficiency.

C7-naphthylethynylated 8-aza-7-deaza-2′-deoxyguanosine naG was synthesized and its photophysical properties were examined. The fluorescent nucleoside exhibited solvatofluorochromic properties (Δλflmax = 67 nm). An ODN probe containing naG forms a stable base pair only with C and discriminates structural changes such as mismatches and deletions by a distinct change in its emission wavelength.

Pyrene-labeled 3-deaza-2′-deoxyadenosine comprising a non-π-conjugated linker py3zA (1) was synthesized and its photophysical properties were investigated. Oligodeoxynucleotide (ODN) probes containing py3zA (1) exhibited remarkable fluorescence quenching only when the opposite base of the complementary strand was the perfectly matched thymine. Such fluorescence quenching-based ODN probes exhibited excellent on-off switching properties, making them useful tools for single nucleotide polymorphism (SNP) genotyping and for the identification of target genes and structural studies of nucleic acids.

8-Aza-3,7-dideaza-2′-deoxyadenosine 1 and its C3-naphthylethynylated derivative 3n7zA (2) comprising a 8-aza-3,7-dideazapurine (pyrazolo[4,3-c]pyridine) skeleton were synthesized for the first time. In particular, nucleoside 3n7zA (2) exhibited environmentally sensitive intramolecular charge transfer (ICT) emission because of electron transition in the coplanar conformer formed by nucleobase and naphthalene moieties. Its incorporation into oligodeoxynucleotide (ODN) probes enable a clear identification of a perfectly matched thymine (T) in the complementary strand by a distinct change in the emission wavelength. In addition, the fluorescence emission of the duplexes containing a cytosine/guanine (C/G) base pair flanking 3n7zA (2) was strongly quenched by guanine only when the opposite base of the modified nucleoside was mismatched, enhancing its base identification ability. Thus, ODN probes containing 3n7zA (2) acted as effective reporter probes for homogeneous single nucleotide polymorphism (SNP) typing.

A novel fluorescent benzo[g]imidazo[4,5-c]quinoline nucleoside BIQA (1) comprising a 3-deaza-2′-deoxyadenosine skeleton was developed and used to monitor BIQA–C base-pair formation in oligodeoxynucleotide (ODN) duplexes. The newly synthesized BIQA exhibited distinct photophysical properties associated with its protonated/deprotonated forms (monomer: pKa 6.2) via dramatic changes in its absorption and fluorescence spectra. In ODN duplexes, the induced protonation of BIQA occurred, even under alkalescent conditions when cytosine was the opposite base on the complementary strand; the resulting BIQA–C base pairs were stable. By monitoring the protonation of BIQA under neutral and alkalescent conditions, we could clearly discriminate cytosine through spectral changes in absorption and fluorescence. Similarly, we found that the demonstrated 3-deaza-2′-deoxyadenosine 3zA forms a stable base pair with cytosine via N1 protonation in ODN duplexes under neutral and acidic conditions (pH < 7.0). At lower pH values, 3zA-containing ODNs could clearly discriminate cytosine through melting temperature (Tm) measurements. Therefore, ODN probes containing indicator nucleosides, such as BIQA and 3zA, exhibit great potential as bioprobes for genetic analysis and structural studies of nucleic acids.

A novel environmentally sensitive fluorescent (ESF) purine nucleoside, cnaA, was synthesized and its photophysical properties were investigated. The base-modified fluorescent nucleoside cnaA exhibited remarkable solvatochromicity and environmentally sensitive dual fluorescence. By using cnaA, we have developed highly thymine (T) selective fluorescent DNA probes that can sense the corresponding T in a target DNA/RNA sequence, as measured by a distinct change in emission wavelength.

G-quenched MBs are devised from readily available starting materials and used for sequence specific DNA detection with high efficiency.

A novel FRET based strategy for DNA sequence analysis utilising base-discriminating fluorescence (BDF) nucleoside, PyU/2-AntU, as donor in the dual-labelled oligonucleotide probe is reported; a selective/specific emission from acceptor, was observed upon excitation at the donor, only when the opposite base of the “smart” fluorescently labeled BDF nucleoside, PyU/2-AntU, is adenine on the complementary target sequence.