Fumie Takei

Screw-Sense-Selective Polymerization of Aryl Isocyanides Initiated by a Pd-Ptμ-Ethynediyl Dinuclear Complex: A Novel Method for the Synthesis of Single-Handed Helical Poly(isocyanide)s with the Block Copolymerization Technique

Living polymerization of chiral aryl isocyanides, such as m- and p-menthoxycarbonylphenyl isocyanides 2 and 5, initiated by the Pd-Pt mu-ethynediyl dinuclear complex 1, proceeds with a high screw-sense selectivity to give the poly(isocyanide)s 3 and 6, which exhibit a large specific rotation and an intense CD band at lambda = 364 nm as a consequence of a helical chirality. The molar optical rotation and molar circular dichroism of the resulting polymers 3 and 6 reach a constant value at a degree of polymerization (Pn) of more than 30. Screw-sense-selective polymerization of achiral aryl isocyanides that bear very bulky substituents, such as 3,5-di(propoxycarbonyl)phenyl isocyanide (11), 3,5-di(butoxycarbonyl)phenyl isocyanide (13), and 3,5-di(cyclohexyloxycarbonyl)phenyl isocyanide (15), is achieved by the use of chiral oligomer complexes 3(30) and 6(30), prepared from the reaction of 1 with 30 equivalents of 2 or 5, as an initiator to give predominantly single-handed helical polymers. In contrast, smaller aryl isocyanides are also polymerized by 3(30) and 6(30) with screw-sense selectivity in the initial stage of the reaction, but the single-handed helix is not preserved up to high molecular weight. Kinetic studies of the polymerization of (L)- and (D)-2, or (L)- and (D)-5 with chiral oligomer complexes (L)-3(50) or (L)-6(100) suggests that the screw sense of the polymer backbone is not controlled kinetically, but rather that the thermodynamically stable screw sense is produced.

Synthesis of organometallic dendrimers with a backbone composed of platinum-acetylide units

Abstract Novel organometallic dendrimers containing platinum-acetylide units in the main chain have been synthesized up to a henicosanuclear complex (12) using triethynylmesitylene (4) as a bridging ligand. The molecular structure of trinuclear complex (5b), which is a model core of the dendrimer, has been established by an X-ray analysis.

Synthesis and redox properties of trinuclear ruthenium–acetylide complexes with tri(ethynylphenyl)amine bridge

Novel trinuclear ruthenium complexes have been prepared by using tri(4-ethynylphenyl)amine as a bridging ligand. Cyclic voltammetry of the trinuclear ruthenium complexes revealed stepwise quasi-reversible redox behavior of three ruthenium-acetylide species and the central triphenylamine unit, whereas the mononuclear analog showed two sequential quasi-reversible redox waves. The spectroelectrochemical UV-VIS spectral studies suggested that the 1e- oxidized triruthenium species was stable and showed a characteristic absorption at lambda(max) = 505 nm. Chemical oxidation of the triruthenium complex with ferrocenium hexafluorophosphate led to the isolation of the 1e- oxidized complex, the near-IR spectrum of which revealed an intervalence charge transfer band due to the electronic coupling among three ruthenium species. The 1e(-) oxidized triruthenium complexes can be classified as class II mixed-valence compounds.

Platinum–acetylide dendrimers possessing a porphyrin core

Intramolecular energy transfer from platinum–acetylide moieties to the porphyrin core was observed in novel organometallic dendrimers that were prepared from a tetra(4-ethynylphenyl)porphyrin-bridged tetranuclear platinum–acetylide core and platinum–acetylide dendrons by a convergent method.

Secondary‐Structure‐Inducible Ligand Fluorescence Coupled with PCR

Single-stranded DNA can change its structure dynamically in response to the presence of a complementary strand. The large structural change from the hairpin secondary structure to the double-stranded form leads to the chemistry of molecular beacons (MBs), or hairpin DNA oligomers having both a fluorescent and a quenching chromophore in the hairpin stem. MBs can be used to report the presence of complementary strands by measuring the increasing fluorescence because of the decreasing energy transfer efficiency. Most DNA probes for fluorescent detection are covalently bound to fluorophores to make energy transfer effective. We have focused on an alternative way to link the fluorescence change with the structural changes of DNA that are induced during polymerase chain reaction (PCR) amplification. We describe herein the chemical concept of noncovalent fluorescent DNA labeling by ligand binding to the secondary structure, which allows us to monitor PCR progress by measuring the change in fluorescence emitted from ligand–primer complexes in a homogeneous solution. This concept is not only complementary to that using fluorescent dye bound selectively to PCR product duplexes, but it also expands the possibility of real-time PCR. The concept of DNA labeling by secondary-structureinducible ligand fluorescence is shown in Figure 1. PCR primers are labeled with a hairpin tag containing cytosine bulges (C-bulges). The molecule 2,7-diamino-1,8-naphthyridine (DANP) binds to a C-bulge in its protonated form (DANPH) and emits fluorescence at 430 nm with a 30 nm bathochromic shift from the fluorescence of free, unbound DANP. We hypothesized that, as the PCR progresses, the hairpin tag will dissolve and be transformed into a duplex, thus resulting in the loss of the DANP binding site and a decrease in the fluorescence at 430 nm. Toward this end, we investigated the hairpin tags. These tags should identify the DANP binding site without disturbing the fluorescence efficiency, should not interfere with the PCR, should be transformed effectively into the duplex during PCR, and should be applicable to diverse primers. First, the sequence flanking the C-bulge producing the highest fluorescence intensity was investigated by measuring DANP fluorescence with duplexes having a C-bulge flanked by A–T and T–A base pairs. The G–C and C–G base pairs were omitted from the flanking base pairs because of their quenching of DANP fluorescence. The characteristic spectrum with a broad single peak at 430 nm was obtained for the A_A/TCT sequence. The relative fluorescence intensity at 450 nm for the four C-bulge duplexes and a fully matched duplex are shown in Figure S1 in the Supporting Information. The C-bulge in the A_A/TCT sequence enhanced DANP fluorescence by 7.6-fold compared with the fully matched duplex and by 82-fold compared with free, unbound DANP. We then designed hairpin tags comprising one to three A_A/TCT units separated by three to five base pairs and a T4 hairpin loop (Table 1). The intensity of DANP fluorescence (FO) was markedly dependent on the number of base pairs separating the C-bulge sites in the hairpin tags. HP-2, which Figure 1. An illustration of the concept of DNA labeling by secondarystructure-inducible ligand fluorescence.

A novel DANP-coupled hairpin RT-PCR for rapid detection of Chikungunya virus.

Chikungunya has re-emerged as an important arboviral infection of global health significance. Because of lack of a vaccine and effective treatment, rapid diagnosis plays an important role in early clinical management of patients. In this study, we have developed a novel molecular diagnostic platform that ensures a rapid and cost-effective one-step RT-PCR assay, with high sensitivity and specificity, for the early detection of the Chikungunya virus (CHIKV). It uses 2,7-diamino-1,8-naphthyridine derivative (DANP)-labeled cytosine-bulge hairpin primers to amplify the nsP2 region of the CHIKV genome, followed by measurement of the fluorescence emitted from DANP-primer complexes after PCRs. The detection limit of our assay was 0.01 plaque-forming units per reaction of CHIKV. Furthermore, the HP-nsP2 primers were highly specific in detecting CHIKV, without any cross-reactivity with the panel of RNA viruses validated in this study. The feasibility of the DANP-coupled hairpin RT-PCR for clinical diagnosis was evaluated using clinical serum samples from CHIKV-infected patients, and the specificity and sensitivity were 100% (95% CI, 80.0% to 100%) and 95.5% (95% CI, 75.1% to 99.8%), respectively. These findings confirmed its potential as a point-of-care clinical molecular diagnostic assay for CHIKV in acute-phase patient serum samples.

Helical Chiral Polyisocyanides Possessing Porphyrin Pendants: Determination of Helicity by Exciton‐Coupled Circular Dichroism

Exciton-coupled circular dichroism of the porphyrin Soret band of triblock copolymers prepared from chiral isocyanide monomers and an achiral tetraphenylporphyrin derivative (TPP) provides a novel method for determining the helical sense of poly(aryl isocyanide)s (see the schematic representation of the structure).

Competitive Allele‐Specific Hairpin Primer PCR for Extremely High Allele Discrimination in Typing of Single Nucleotide Polymorphisms

Allele-specific polymerase chain reaction (AS-PCR) is one of the most convenient and direct methods for single nucleotide polymorphism (SNP) typing, and is based on the selective elongation of the primer that matches the template sequence. However, the discrimination of two alleles by allele-specific primers is not always complete because of subtle differences in the thermodynamic stability between matched and mismatched primer-template complexes. Elongation of the primer from the mismatched primer–template complex will eventually match the template to the formerly mismatched primer. Because of the exponential amplification of the analyte DNA by PCR, formation of a matched template for the mismatched primer significantly reduces allele specificity. Although methods to improve allele specificity have been developed by using primer design, allele specific blocking and clamping, 26] and engineered polymerase, a complete solution remains to be devised. Another issue in AS-PCR is the fact that diagnosis with ASPCR usually relies on the detection of amplified double-stranded DNA (dsDNA). Because dsDNA can be produced from both matched and mismatched primer–template complexes, diagnosis with AS-PCR by detecting dsDNA has some intrinsic ambiguity. To address this second issue, we have used primer labeling and have reported on a hairpin primer PCR (HPPCR) method, which monitors the progress of PCR by detecting not the producing dsDNA but the amount of primer consumed (or remaining). In the HP-PCR method, the primers are labeled with a hairpin tag containing a cytosine bulge (Cbulge). The fluorescent molecule 2,7-diamino-1,8-naphthyridine (DANP; Scheme 1 A) bound to the C-bulge and emitted fluorescence at around 430–450 nm with a 30 nm bathochromic shift from the fluorescence of free, unbound DANP. The hairpin structure was unfolded and converted into the double-stranded form by DNA polymerase with concomitant release of the bound DANP from the hairpin tag, resulting in a decrease in the fluorescence at 450 nm. The fluorescence intensity was monitored at 450 nm to quantify consumption of the HP in this HP-PCR method. 35] Exploiting this ability of HP-PCR to detect the amount of primer, as compared with conventional PCR, which detects the product dsDNA, we here describe our approach to solve the first issue of AS-PCR by using competitive allele-specific HPPCR. Using a competitive primer in HP-PCR, we have achieved extremely high allele specificity in AS-PCR. Competitive allele-specific HP-PCR uses two kinds of forward primer simultaneously: one with a hairpin tag (hairpin primer, HP) and the other without a hairpin tag (competitor primer, CP), which are allele-specific in terms of the nucleotide base at the SNP site (Scheme 1 B and C). When the nucleotide base of the analyte DNA at the SNP site matched that of the HP, polymerase selectively elongated the HP, eventually leading to a decrease in the fluorescence intensity by unfolding the hairpin tag and releasing the bound DANP. The CP that is mismatched to the analyte DNA had virtually no effect on the PCR (Scheme 1 B). When the HP did not match the template, we anticipated that the elongation of the HP on the mismatched template would be effectively suppressed by the formation of a matched CP–template complex (Scheme 1 C). To prove our concept of competitive allele-specific HP-PCR, we investigated the typing of a G/T SNP embedded at nucleoScheme 1. Illustration of competitive allele-specific HP-PCR. A) Chemical structure of DANP and hydrogen bonding to a cytosine bulge with protonation. B) Selective elongation of the HP on the matched template. C) Suppression of elongation of the HP on the mismatched template by competitor primer.

Polymerization of aryl isocyanides by cyclopentadienylnickel–alkynyl complexes

Abstract ( η 5 -Cyclopentadienyl)nickel–alkynyl complexes, CpNi(PPh 3 )CCR (R=Ph, H, CO 2 Et), and -methyl complex effectively catalyze the polymerization of aryl isocyanides at room temperature. Double-insertion complexes of isocyanides are isolated and characterized as an active intermediate of the polymerization, whose mechanism involving successive insertion of isocyanides into nickel–carbon σ -bonds is proposed.

Synthesis and reaction of DNA oligomers containing modified cytosines related to bisulfite sequencing.

The synthesis of DNA oligomers containing N(4)-hydroxy-5,6-dihydrocytosine-6-sulfonate by using ligand-induced base flipping of cytosine followed by the simultaneous addition of bisulfite and hydroxylamine is reported. In contrast to C, the flipped-out 5-methylcytosine was selectively oxidized over thymines and cytosines in the duplex by potassium permanganate. Ligand-induced base flipping is a convenient and powerful strategy for the synthesis of modified cytosines and 5-methylcytosines related to bisulfite sequencing at the predetermined site of DNA.