Shuhei Kusano

Enantioselective Synthesis of 3-Arylquinazolin-4(3H)-ones via Peptide-Catalyzed Atroposelective Bromination

We report the development of a tertiary amine-containing β-turn peptide that catalyzes the atroposelective bromination of pharmaceutically relevant 3-arylquinazolin-4(3H)-ones (quinazolinones) with high levels of enantioinduction over a broad substrate scope. The structure of the free catalyst and the peptide-substrate complex were explored using X-ray crystallography and 2D-NOESY experiments. Quinazolinone rotational barriers about the chiral anilide axis were also studied using density functional theory calculations and are discussed in light of the high enantioselectivities observed. Mechanistic studies also suggest that the initial bromination event is stereodetermining, and the major monobromide intermediate is an atropisomerically stable, mono-ortho-substituted isomer. The observation of stereoisomerically stable monobromides stimulated the conversion of the tribromide products to other atropisomerically defined products of interest. For example, (1) a dehalogenation Suzuki-Miyaura cross-coupling sequence delivers ortho-arylated derivatives, and (2) a regioselective Buchwald-Hartwig amination procedure installs para-amine functionality. Stereochemical information was retained during these subsequent transformations.

Formation of highly selective and efficient interstrand cross-linking to thymine without photo-irradiation

Interstrand cross-linking (ICL) forming oligodeoxynucleotides (ODNs) have been expected to ensure the inhibition of gene expression. In this communication, we report a highly efficient and selective ICL reaction to thymine using a 4-amino-2-vinyl-6-oxopyrimidine derivative.

Production of truncated protein by the crosslink formation of mRNA with 2'-OMe oligoribonucleotide containing 2-amino-6-vinylpurine.

The development of convenient methods for controlling the protein expression is an important challenge in the postgenomic era. We applied the crosslink forming oligonucleotide (CFO) as a terminator of the ribosomal translation. In this study, we demonstrated that the improved reactivity of our CFO under physiological conditions enabled the sequence-specific introduction of a steric block for a ribosome on mRNAs. In vitro and in cell translation experiments revealed that the crosslinked mRNA can produce the truncated proteins in which the translation terminates at the desired position.

Crosslinking reactions of 4-amino-6-oxo-2-vinylpyrimidine with guanine derivatives and structural analysis of the adducts

DNA interstrand crosslinks (ICLs) are the primary mechanism for the cytotoxic activity of many clinical anticancer drugs, and numerous strategies for forming ICLs have been developed. One such method is using crosslink-forming oligonucleotides (CFOs). In this study, we designed a 4-amino-6-oxo-2-vinylpyrimidine (AOVP) derivative with an acyclic spacer to react selectively with guanine. The AOVP CFO exhibited selective crosslinking reactivity with guanine and thymine in DNA, and with guanine in RNA. These crosslinking reactions with guanine were accelerated in the presence of CoCl2, NiCl2, ZnCl2 and MnCl2. In addition, we demonstrated that the AOVP CFO was reactive toward 8-oxoguanine opposite AOVP in the duplex DNA. The structural analysis of each guanine and 8-oxoguanine adduct in the duplex DNA was investigated by high-resolution NMR. The results suggested that AOVP reacts at the N2 amine in guanine and at the N1 or N2 amines in 8-oxoguanine in the duplex DNA. This study demonstrated the first direct determination of the adduct structure in duplex DNA without enzyme digestion.

Biotinylated Cyclophane: Synthesis, Cyclophane-Avidin Conjugates, and Their Enhanced Guest-Binding Affinity.

Cationic and anionic cyclophanes bearing a biotin moiety were synthesized as a water-soluble host (1a and 1b, respectively). Both hosts 1a and 1b were found to strongly bind avidin with binding constants of 1.3 × 10(8) M(-1), as confirmed by surface plasmon resonance measurements. The present conjugate of 1a with avidin (1a-avidin) showed an enhanced guest binding affinity toward fluorescence guests such as TNS and 2,6-ANS. The K values of 1a-avidin conjugate with TNS and 2,6-ANS were ~19-fold larger than those of monocyclic cyclophane 1a with the identical guests. Favorable hydrophobic and electrostatic interactions between 1a-avidin and TNS were suggested by computer-aided molecular modeling calculations. Moreover, addition of excess biotin to the complexes of 1a-avidin with the guests resulted in dissociation of 1a-avidin to avidin and 1a having less guest-binding affinity. Conversely, such enhancements in the guest-binding affinity were not obviously observed for the conjugate of anionic 1b with avidin (1b-avidin) due to electrostatic repulsion between anionic 1b and anionic guests.

Synthesis of Water-Soluble Cyclophane Hexamers Having a Triphenylene Core and Their Enhanced Guest-Binding Behavior

A key compound, a precursor of water-soluble cyclophane hexamer, was prepared via Williamson ether synthesis of tetraaza[6.1.6.1]paracyclophane derivatives bearing a bromoacetamide moiety with triphenylene-2,3,6,7,10,11-hexaol as a core. A cationic cyclophane hexamer (1) was obtained by removing the protecting groups from the precursor. Fluorescence titration experiments proved that cationic cyclophane hexamer 1 showed macrocyclic multivalency effects; i.e., 1:1 host/guest binding constants (K) of 1 with anionic guests, 6-anilinonaphthalene-2-sulfonate and 6-p-toluidinonaphthalene-2-sulfonate, were increased about 63- and 62-fold, respectively, relative to those of monomeric cyclophane. Similarly, anionic cyclophane hexamer 2, which was easily prepared from 1, showed macrocyclic multivalency effects in K values with cationic guests such as hydrochlorides of doxorubicin and daunorubicin as an anticancer drug.

Synthesis of 6-amino-2-vinylpurine derivatives for cross-linking and evaluation of the reactivity.

Oligodeoxynucleotides (ODNs) have been widely used for inhibiting the gene expression in antisense or antigene methods, and the interstrand cross-linking (ICL) forming ODNs have been expected to ensure the inhibition by these methods. Previously, we reported a highly efficient and selective ICL reaction toward cytosine using the 2-amino-6-vinylpurine derivative under acidic conditions. In this Letter, we report the synthesis of ODN containing 6-amino-2-vinylpurine derivatives and evaluation of the cross-linking reactivity.

Design and synthesis of the novel cross-linking agent

Previously, we have developed a highly efficient and selective cross-linking reaction to the cytosine base at the target site of DNA using the oligodeoxynucleotide (ODN) containing 2-amino-6-vinylpurine derivative (1). Based on these results, we have designed the novel cross-linking agents, which are pyrimidine derivatives having two hydrogen bond sites and vinyl group as a reactive moiety. In this paper, we wish to report the results to investigate on the synthesis of the pyrimidine derivatives having potential as novel cross-linking agents.

Synthesis of a cysteine-linked cyclophane dimer having two rhodamine moieties and its reduction-responsive degradation as studied by fluorescence spectroscopy

Cysteine-linked cyclophane dimer having two rhodamine moieties (2) was synthesized as a reduction-responsive host. Owing to self-quenching property of the two rhodamine moieties, cyclophane dimer 2 showed weak fluorescence intensity relative to that of the rhodamine B moiety itself. The cleavage of disulfide bond of 2 was performed by a treatment with reducing agents such as dithiothreitol, to give the corresponding monomeric cyclophanes having a rhodamine moiety. Such reductive degradation of 2 was detected by the increase on fluorescence intensity. As a host, cyclophane dimer 2 was found to show a stronger guest-binding affinity than the monomeric cyclophanes due to concentration effects of the macrocycles. In addition, reduction-responsive release of entrapped guest molecules by 2 was also monitored by fluorescence spectroscopy.

Synthesis and effect of linker length on guest-binding affinity of water-soluble tetraazacyclophane dimers

Cationic cyclophane dimers which are constructed with two tetraaza[6.1.6.1]para-cyclophanes and a short or long hydrophilic linker interposed between them were synthesized as a water-soluble host (1a and 1b, respectively). The binding constant (K) values of 1a having a short linkage with fluorescence guests such as 6-p-toluidinonaphthalene-2-sulfonate and 6-anilinonaphthalene-2-sulfonate were about 2-fold larger than those of 1b having a long one with the identical guests. Local concentration in the macrocycles of 1a was more effective than that of 1b for such enhancements of the guest-binding affinity. Moreover, the K values of cationic cyclophane dimer having a short hydrophobic linkage 1c were comparable to those by 1a, indicating that guest-binding affinity was less affected on the hydrophilic/hydrophobic characteristics of these short linkers.