Kazuhiko Nakatani

Scanning of guanine-guanine mismatches in DNA by synthetic ligands using surface plasmon resonance

Here we have designed and synthesized ligands that specifically bind with high affinity (Kd = 53 nM) to the guanine (G)–guanine mismatch, one of four types of single-nucleotide polymorphism (SNP). Detection of the G-G mismatch was performed by a surface plasmon resonance (SPR) assay using a sensor chip carrying the G-G specific ligand on its surface. The accuracy of the G-G mismatch detection by the SPR sensor was demonstrated by a marked SPR response obtained only for the DNA containing the G-G mismatch. DNAs containing G-A and G-T mismatches, as well as a fully matched duplex, produced only a weak response. Furthermore, this assay was found applicable for the detection of SNP existing in PCR amplification products of a 652-nucleotide sequence of the HSP70-2 gene.

Chemistry Challenges in SNP Typing

Single nucleotide polymorphisms (SNPs) are gene variations that result from a single nucleotide difference. A single Watson–Crick base pair in wild-type DNA is replaced by any one of the other three base pairs in mutant DNA. When an SNP site is located in a protein-encoding region, the amino acid sequence of the produced protein may have a chance to be changed. SNPs in a promoter region may modulate the transcriptional efficiency of proteins encoded downstream. As a consequence, a small difference in DNA sequence can result in a much larger difference in the phenotype of each individual. SNP typing that identifies the base at predetermined polymorphic sites in any given DNA sample is envisioned as an essential technology in the near feature for realizing personalized medicine. Several methods for SNP typing have been studied, and some of them have already become commercially available. Because there is only a one-base-pair difference between the mutant and wild-type DNAs, it is difficult to differentiate the two DNAs. Most current SNP-typing techniques are indebted to the high fidelity of DNA polymerase in primer extension and flap endonuclease in the digestion reaction of a specific DNA structure. While the difference between the two DNAs is only subtle, increasing knowledge and improving technologies in chemistry and associated fields should lead to a conceptually new solution in SNP typing. Developing nonenzymatic methods for SNP typing offers great opportunities and challenges in chemistry. Among the numerous reports on new methods of SNP typing, this minireview focuses primarily on the chemistry basis of newly developed and developing technologies for SNP typing that do not make use of enzymes, except for PCR amplification. The methods of nonenzymatic SNP typing described here show only the tip of the large number of studies that have appeared in recent scientific journals. Readers who are interested in general methods of SNP typing should consult the comprehensive reviews. The SNP typing strategies described in this review were compared to the principles for allele discrimination, detection signals, and detection platforms (Table 1). While there is no standard condition to compare the sensitivity, accuracy, and performance of each typing method with the others, the amounts of genome, PCR products, or oligonucleotides described for demonstrating the performance of the nonenzymatic methods in the literature are briefly mentioned in Table 1.

Improved selectivity for the binding of naphthyridine dimer to guanine-guanine mismatch.

Naphthyridine dimer composed of two 2-amino-1,8-naphthyridines and a connecting linker strongly binds to guanine-guanine (G-G) mismatch in duplex DNA. In order to improve G-G selectivity for the binding, we have examined structure modification of the linker. A new naphthyridine dimer possessing 3,6-diazaoctanedioic acid linker binds to G-G mismatch with an association constant of 1.18 x 10(7) M(-1), which is somewhat weaker than that of the original naphthyridine dimer having a shorter connecting linker. However, the binding of the modified naphthyridine dimer to G-A mismatch was almost negligible as compared to that of the original. This results in a net increase of the selectivity for the binding to G-G mismatch by 4-folds.

Ligand Binding to Tandem G Quadruplexes from Human Telomeric DNA

Ligand-induced stabilization of intramolecular telomeric G quadruplexes produced in the single-stranded overhang of the human telomere has become an attractive strategy for the development of anticancer drugs. Several distinct solution conformations of human telomeric G quadruplexes have been elucidated in the presence of sodium and potassium cations. The K-form, hybrid-type G-quadruplex structure has been considered to be a physiologically relevant conformation of the human telomeric sequence, and thus, can be specifically targeted by G-quadruplex-interactive, small-molecule ACHTUNGTRENNUNGdrugs. Recently, a beads-on-a-string model was proposed for the telomeric overhang, in which every four consecutive Grich repeats adopt an individual G-quadruplex structure, and two G-quadruplex units are connected by one TTA linker. c, 4] Ligand binding to the G quadruplex has mostly been investigated on telomere sequences producing a single G quadruplex, but few studies of ligand binding to beads-on-a-string G quadruplexes have been reported. To gain insight into the beads-on-a-string model and the nature of ligand binding, we undertook the polymerase stop assay on human telomere sequences of three to eight repeats (Table S1 in the Supporting Information) with TMPyP4, a G-quadruplex-interactive ligand, and sanguinarine (Scheme 1), a natural isoquinoline alkaloid. The results described in this paper confirm the beads-on-astring structure of telomeric overhang and suggest a mode of ligand binding between tandem G-quadruplex beads. These observations should be taken into account for structure-based design of anticancer drugs targeting human telomeric DNA. Sanguinarine is a natural isoquinoline alkaloid isolated from the North American herb bloodroot (Sanguinaria canadensis). It was approved by the FDA in 2003 to be added to oral cleansing products as an antibacterial agent. Sanguinarine also possesses potent anticancer activity. We have previously reported its DNA-binding activity and distinct sequence selectivity to double-stranded DNA, which was proposed to be one of the molecular mechanisms of its anticancer activity. The structural similarity of sanguinarine with berberine, another isoquinoline alkaloid possessing G-quadruplex-binding activity, prompted us to speculate that sanguinarine is probably a G-quadruplex binder. In this communication, we report its binding to the ACHTUNGTRENNUNGtelomeric overhang using DNA polymerase stop assays. DNA templates Tem-3 and Tem-4 (Table S1), which contains three and four human telomeric repeats d ACHTUNGTRENNUNG(TTAGGG), respectively, were employed, and TMPyP4 was used as the reference compound in the assay. Neither TMPyP4 nor sanguinarine blocked DNA synthesis on Tem-3, because an intramolecular Gquadruplex structure could not form with three human telomeric repeats on Tem-3 (Figure 1). In contrast, both sanguinarine and TMPyP4 produced paused bands in DNA synthesis on Tem-4. The position of the paused bands was the same for the two ligands. For each ligand, a series of concentration-dependent paused bands appeared at the beginning of the G-quadruplex-forming site, that is, the first site of G-rich repeats in Tem-4 (from 3’ to 5’). In the presence of 3 mm TMPyP4, the polymerase reaction was totally suppressed to give no elongation of the primer. The tight binding of sanguinarine and TMPyP4 to the K-form hybrid-type G-quadruplex structure was clearly indicated from the large increase in the melting temperature (DTm) of dAGGG ACHTUNGTRENNUNG(TTAGGG)3 (11.4 8C for sanguinarine and 9.5 8C for TMPyP4 in a 2:1 ligand/DNA molar ratio). ACHTUNGTRENNUNGFurthermore, a broad negative induced band at 351 nm in the CD spectra confirmed the interaction between sanguinarine and the K-form G quadruplex formed by dAGGG ACHTUNGTRENNUNG(TTAGGG)3 (Figure 2). Regarding the mode of ligand binding to the G quadruplex, NMR, 10a,11] fibre diffraction, and single-crystal X-ray crystallographic analyses have confirmed that a variety of planar aromatic binders including TMPyP4 interact with the G quadruplex by end stacking rather than intercalation between G tetrads. TMPyP4 was also found to stack onto the external loop of a bimolecular G quadruplex in a crystal structure. Recently, electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) has been used to analyze complexes of G quadruplexes with ligands, since G quadruplexes can maintain their structure in the gas phase in the presence of a suitable cation such as NH4 + . ESI-MS showed the ammonium ion to be loScheme 1. Chemical structures of sanguinarine and TMPyP4.

Highly efficient photochemical generation of o-Quinone methide from Mannich bases of phenol derivatives

Abstract o -Quinone methide was efficiently produced by photoirradiation of phenolic Mannich bases in aqueous acetonitrile with low energy UV light. The photoreaction proceeds most effectively at neutral pH in aqueous solvents.

Control of DNA hybridization by photoswitchable molecular glue.

Hybridization of DNA is one of the most intriguing events in molecular recognition and is essential for living matter to inherit life beyond generations. In addition to the function of DNA as genetic material, DNA hybridization is a key to control the function of DNA-based materials in nanoscience. Since the hybridization of two single stranded DNAs is a thermodynamically favorable process, dissociation of the once formed DNA duplex is normally unattainable under isothermal conditions. As the progress of DNA-based nanoscience, methodology to control the DNA hybridization process has become increasingly important. Besides many reports using the chemically modified DNA for the regulation of hybridization, we focused our attention on the use of a small ligand as the molecular glue for the DNA. In 2001, we reported the first designed molecule that strongly and specifically bound to the mismatched base pairs in double stranded DNA. Further studies on the mismatch binding molecules provided us a key discovery of a novel mode of the binding of a mismatch binding ligand that induced the base flipping. With these findings we proposed the concept of molecular glue for DNA for the unidirectional control of DNA hybridization and, eventually photoswitchable molecular glue for DNA, which enabled the bidirectional control of hybridization under photoirradiation. In this tutorial review, we describe in detail how we integrated the mismatch binding ligand into photoswitchable molecular glue for DNA, and the application and perspective in DNA-based nanoscience.

Synthetic studies on duocarmycin. 1. Total synthesis of dl-duocarmycin A and its 2-epimer

Abstract The title synthesis was first achieved by employing novel methoxycarbonylation of the C4-position of the 5-aminoindoline by way of the isatin and subsequent Dieckmann cyclization to the methyl 2-methylindoxyl-2-carboxylate as key steps. In vitro cytotoxicity assay against P388 murine leukemia obviously disclosed that cytotoxicities of the synthesized compounds are comparable and almost half of that of natural (+)-duocarmycin A.

Synthesis and cytotoxicity of the acyclic (E)- and (Z)-dienediyne systems related to neocarzinostatine chromophore

Abstract Synthesis of the title compounds could be achieved by featuring the Pd-catalyzed coupling reaction of stereo-defined (E)-and (Z)-enol triflates with an acetylene derivative. It was found that the acyclic (Z)-dienediyne system obviously exhibits in vitro cytotoxicity against P388 murine leukemia stronger than that for the corresponding (E)-isomer.

DNA alkylation properties of the duocarmycins: (+)-duocarmycin A, epi-(+)-duocarmycin A, ent-(−)-duocarmycin A and epi,ent-(−)-duocarmycin A

Abstract A study of the comparative in vitro cytotoxic activity and DNA alkylation properties of both enantiomers of the two diastereomers of (+)-duocarmycin A are detailed. The DNA alkylation efficiency and in vitro cytotoxic potency of the natural enantiomers ((+)-duocarmycin A epi -(+)-duocarmycin A, 3-8x) exceed those of the unnatural enantiomers ( ent -(−)-duocarmycin A, epi,ent -(−)-duocarmycin A) by at least 100x.

Oxidative fragmentation of γ-hydroxyalkyl stannanes stereospecific formation of (E) and (Z)-keto olefins

Abstract Treatment of γ-hydroxyalkyl stannanes with lead tetraacetate in refluxing benzene leads to the stereospecific formation of (E) and (Z)-keto olefins according to the stereochemistry of the starting materials in excellent yield.