Chiral Assemblies of Planar and Achiral meta-Arylene Ethynylene Macrocycles Induced by Saccharide Recognition.

We created chiral assemblies of planar and achiral macrocycles by saccharide recognition. To achieve this, we synthesized stackable meta-arylene ethynylene macrocycles consisting of pyridine-acetylene-phenol and pyridine-acetylene-aniline units. 1H NMR, absorption, and fluorescence emission spectroscopy indicated that these macrocycles formed 1:1 and 2:1 complexes with lipophilic alkyl glycosides. The 2:1 complex of the pyridine-acetylene-phenol macrocycle showed induced circular dichroism (ICD) bands, meaning that two achiral macrocycles are arranged in an asymmetrically twisted manner. CD spectroscopy revealed that the helical sense was affected by the chirality of guest saccharides. On the other hand, strong CD bands were observed after solid-liquid extraction of native saccharides into lipophilic solvents using the pyridine-acetylene-aniline macrocycle.

2-Aminopyridine as a Nucleobase Substitute for Adenine in DNA-like Architectures: Synthesis of Alkynyl C-Nucleotides and Their Hybridization Characteristics.

Halogenated 2-aminopyridine was attached to the acetylene terminal of ethynyl C-2-deoxy-ß-d-ribofuranoside as a nucleobase substitute, and then, the C-nucleoside was incorporated into natural DNAs. The resulting chimeric DNA constructed double helical structures with the complementary chimeric DNA. In the duplex, 2-aminopyridine functioned as an adenine analogue that formed a base pair with a non-natural thymine isostere. Artificial homooligomers were also prepared only from the adenine-type C-nucleoside and proven to form completely artificial double helices with the corresponding artificial thymine-type homooligomers.

Additive-Free Enzymatic Phosphorylation and Ligation of Artificial Oligonucleotides with C-Nucleosides at the Reaction Points.

We report enzymatic phosphorylation and additive-free ligation of DNAs containing unnatural C-nucleotide residues through the action of T4 polynucleotide kinase and T4 DNA ligase. The artificial units are each made up of an alkynyl deoxyribose component and one of the unnatural nucleobases D*, T*, G*, and C*, corresponding-from a viewpoint of hydrogen-bonding patterns-to natural A, T, G, and C, respectively. Phosphorylation progressed quantitatively at the 5'-end in the cases of all of the artificial units in the chimeric DNAs. Ligation also smoothly progressed at the 5'-end in the cases of the D* and G* nucleotide residues, but only negligibly in those of their T* and C* counterparts. Chemical redesign of the last two units successfully improved the ligation efficiency, so that enzymatic ligation worked well for all of the artificial units in every 3'-natural⋅5'-artificial, 3'-artificial⋅5'-natural, and 3'-artificial⋅5'-artificial terminal combination at the nicks.

Photoinduced Electron Transfer-Regulated Protein Labeling With a Coumarin-Based Multifunctional Photocrosslinker.

We developed a novel diazirine-based photolabeling agent having a (coumarin-4-yl)methyl ester scaffold, which exhibited multiple photochemical properties of crosslinking, fluorogenicity and cleavage. These properties can be kinetically regulated via photoinduced electron transfer between diazirine and coumarin moieties. The C-O bond of (coumarin-4-yl)methyl ester can be cleaved via photochemical excitation of coumarin moiety, that function has been initially quenched by the diazirine moiety. Upon diazirine photolysis with 365-nm light, interacting protein was stably captured with photoactivatable ligand probe. Then, the unlocked cleavage function was activated with 313 nm light, and the reaction was accelerated in a weakly-basic solution. The crosslinked protein could be selectively isolated with attachment of a small coumarin tag on the surface. This multi-functional labeling agent has a great potential to facilitate LC-MS/MS-based protein identification.

Syndecan-4 Is a Receptor for Clathrin-Mediated Endocytosis of Arginine-Rich Cell-Penetrating Peptides.

Arginine-rich cell-penetrating peptides (CPPs) such as Tat and oligoarginine peptides have been widely used as carriers for intracellular delivery of bioactive molecules. Despite accumulating evidence for involvement of endocytosis in the cellular uptake of arginine-rich CPPs, the primary cell-surface receptors for these peptide carriers that would initiate endocytic processes leading to intracellular delivery of bioactive cargoes have remained poorly understood. Our previous attempt to identify membrane receptors for octa-arginine (R8) peptide, one of the representative arginine-rich CPPs, using the photo-cross-linking probe bearing a photoreactive diazirine was not successful due to considerable amounts of cellular proteins nonspecifically bound to the affinity beads. To address this issue, here we developed a photo-cross-linking probe in which a cleavable linker of a diazobenzene moiety was employed to allow selective elution of cross-linked proteins by reducing agent-mediated cleavage. We demonstrated that introduction of the diazobenzene moiety into the photoaffinity probe enables efficient purification of cross-linked proteins with significant reduction of nonspecific binding proteins, leading to successful identification of 17 membrane-associated proteins that would interact with R8 peptide. RNAi-mediated knockdown experiments in combination with the pharmacological inhibitors revealed that, among the proteins identified, syndecan-4, one of the heparan sulfate proteoglycans, is an endogenous membrane-associated receptor for the cellular uptake of R8 peptide via clathrin-mediated endocytosis. This syndecan-4-dependent pathway was also involved in the intracellular delivery of bioactive proteins mediated by R8 peptide. These results reveal that syndecan-4 is a primary cell-surface target for R8 peptide that allows intracellular delivery of bioactive cargo molecules via clathrin-mediated endocytosis.

Synthesis of Nonnatural Oligonucleotides Made Exclusively of Alkynyl C-Nucleosides with Nonnatural Bases.

This unit describes detailed procedures for the preparation of nonnatural C-nucleosides comprising seven types of nonnatural nucleobases attached to 1'-position of 2'-deoxyribose through an acetylene bond with the ß-configuration. In addition, derivatization of these alkynyl C-nucleosides into the corresponding phosphoramidites and the subsequent oligonucleotide synthesis are also presented. The processes are depicted in three parts. The first basic protocol deals with the synthesis of a key intermediate, 5-O-DMTr-protected 1-ethynyl-2-deoxy-ß-D-ribofuranoside. The second basic protocol mentions the procedures of the preparation of nonnatural C-nucleosides by a palladium-catalyzed coupling reaction of the ethynyl intermediate and halogen-attached nonnatural nucleobases. The synthetic procedures of the corresponding nonnatural phosporamidites are also described. The third basic protocol presents the solid-phase, automated synthesis of nonnatural oligonucleotides composed exclusively of the nonnatural C-nucleotides.

A firmly hybridizable, DNA-like architecture with DAD/ADA- and ADD/DAA-type nonnatural base pairs as an extracellular genetic candidate.

We describe artificial DNA molecules exclusively consisting of four types of alkynyl C-nucleotides with nonnatural bases. The artificial DNA exhibited almost the same characteristics as natural DNA, such as in regard to the stepwise duplex and triplex formation and the right-handed higher-order structure with an antiparallel alignment fashion.

An isotope-coded fluorogenic cross-linker for high-performance target identification based on photoaffinity labeling.

A photoaffinity labeling (PAL)-based method for the rapid identification of target proteins is presented in which a high-performance chemical tag, an isotope-coded fluorescent tag (IsoFT), can be attached to the interacting site by irradiation. Labeled peptides can be easily distinguished among numerous proteolytic digests by sequential detection with highly sensitive fluorescence spectroscopy and mass spectrometry. Subsequent MS/MS analysis provides amino acid sequence information with a higher depth of coverage. The combination of PAL and heterogeneous target-selecting techniques significantly reduces the amount of time and protein required for identification. An additional photocleavable moiety successfully accelerated proteomic analysis using cell lysate. This method is a widely applicable approach for the rapid and accurate identification of interacting proteins.

Coupling reaction of thioamides with sulfonyl azides: an efficient catalyst-free click-type ligation under mild conditions.

We report a coupling reaction of thioamides and sulfonyl azides to generate sulfonyl amidines in the absence of any activation additives. The reaction progresses in various solvents under mild conditions. Water exhibits the highest performance with respect to efficiency.

Identification of cellular proteins interacting with octaarginine (R8) cell-penetrating peptide by photo-crosslinking.

Octaarginine (R8) is a representative cell-penetrating peptide. Lanthionine synthetase component C-like protein 1 (LanCL1) was identified as a potential intracellular target of R8 by using a photo-crosslinking assay that utilized a phenyl-trifluoromethyl diazirine moiety and peptide mass fingerprinting. Increased cellular uptake of R8 by LanCL1-overexpressing cells was observed.

A supramolecular DNA self-assembly based on ß-cyclodextrin-adamantane complexation as a bioorthogonal sticky end motif.

We propose linear end-to-end assemblies of short DNA duplexes based on ß-cyclodextrin-adamantane complexation. The assembled duplexes exhibited increased Tm values compared with those of the corresponding natural hybrids. Competition experiments with external guest molecules showed a substantial decrease in Tm of the terminal modified duplexes, suggesting the viability of inter-duplex complexation.

Electrochemical direct detection of DNA deamination catalyzed by APOBEC3G.

APOBEC3G catalyzes deamination of cytosines in HIV-1 genome, and restricts the HIV-1 infection. Here, we propose a picomole-scale assay for the detection of DNA deamination catalyzed by APOBEC3G. Our results show the suitability of the developed method for a time course analysis of enzyme-catalyzed DNA modifications.

Electrochemical detection of insertion/deletion mutations based on enhanced flexibility of bulge-containing duplexes on electrodes.

Ferrocene-modified DNA probes formed fully matched duplexes and bulge-containing ones with wild-type and insertion/deletion-type complements of clinical importance, respectively. Cyclic voltammetry measurements revealed that the bulge-containing duplexes showed an increased flexibility compared to the fully matched duplexes. The difference in the bending elasticity could be read out electrochemically by square wave voltammetry.

Specific induced circular dichroism and enhanced B to z transitions of duplexes stabilized by chromophore-linked alkynylnucleoside residues.

We have developed an induced circular dichroism (ICD) probe with a chromophore-linked alkynyldeoxyribose skeleton for analyzing higher-order structures of DNA duplexes in the visible-light region. When CG-repeated oligonucleotides (ODNs) with the probe at their 5' ends adopted Z-form duplexes at a high NaCl concentration, strong ICD signals were observed at the absorptive region of the chromophore. On the other hand, their B-form duplexes, formed at a low NaCl concentration, produced a faint ICD signal. The specific ICD for the Z-form duplexes was found to appear only when the chromophores were attached at the 5' ends of each of the ODNs. Furthermore, the chromophoric alkynylnucleoside residues effectively promoted the B to Z transition of the ODN.

Detection of mismatched duplexes by synchronizing the pulse potential frequency with the dynamics of ferrocene/isoquinoline conjugate-connected DNA probes immobilized onto electrodes.

Cyclic voltammetry was performed at various scan rates for the duplexes from ferrocene/isoquinoline conjugate-connected DNA probes on gold electrodes. The relationship between the observed currents and the scan rates disclosed the enhanced bending elasticity of the mismatched duplexes compared with the fully matched duplexes. The difference of the dynamics was easily detected through the currents from the conjugate by adjusting the pulse potential frequency in square-wave voltammetry. By using the present strategy, we succeeded in accurately detecting various naturally occurring single-nucleotide polymorphisms.

Artificial DNA made exclusively of nonnatural C-nucleosides with four types of nonnatural bases.

We describe a new class of DNA-like oligomers made exclusively of nonnatural, stable C-nucleosides. The nucleosides comprise four types of nonnatural bases attached to a deoxyribose through an acetylene bond with the beta-configuration. The artificial DNA forms right-handed duplexes and triplexes with the complementary artificial DNA. The hybridization occurs spontaneously and sequence-selectively, and the resulting duplexes have thermal stabilities very close to those of natural duplexes. The artificial DNA might be applied to a future extracellular genetic system with information storage and amplifiable abilities.

Photooxygenation of alkynylperylenes. Formation of dibenzo[jk,mn]phenanthrene-4,5-diones.

3-(1-Alkynyl)perylenes undergo oxygenation when subjected to irradiation with visible light under aerated conditions. The structures of novel oxygenated products formed in this manner are assigned as regioisomeric dibenzo[jk,mn]phenanthrene-4,5-diones.