Shinya Hagihara

Probing strigolactone receptors in Striga hermonthica with fluorescence

Germination signals illuminated The noxious weed Striga can take down an entire crop. Fields in Africa are particularly susceptible to the devastation it can cause. Striga seeds germinate in response to faint traces of the hormone strigolactone released by its targets. Tsuchiya et al. designed a mimic of strigolactone that, when cleaved by the Striga receptor, generates a fluorescent end product. This photogenic mimic lit up Striga seeds upon germination and led to the identification of its strigolactone receptor. Abolishing the activity of this receptor could be an effective defensive strategy. Science, this issue p. 864 The parasitic weed Striga has given up the secret of its key receptor. Elucidating the signaling mechanism of strigolactones has been the key to controlling the devastating problem caused by the parasitic plant Striga hermonthica. To overcome the genetic intractability that has previously interfered with identification of the strigolactone receptor, we developed a fluorescence turn-on probe, Yoshimulactone Green (YLG), which activates strigolactone signaling and illuminates signal perception by the strigolactone receptors. Here we describe how strigolactones bind to and act via ShHTLs, the diverged family of α/β hydrolase-fold proteins in Striga. Live imaging using YLGs revealed that a dynamic wavelike propagation of strigolactone perception wakes up Striga seeds. We conclude that ShHTLs function as the strigolactone receptors mediating seed germination in Striga. Our findings enable access to strigolactone receptors and observation of the regulatory dynamics for strigolactone signal transduction in Striga.

Boronic Acid Converters for Reactive Hydrazide Amplifiers: Polyphenol Sensing in Green Tea with Synthetic Pores

Multicomponent sensing in complex matrices with synthetic pores became feasible with the introduction of amplifiers. Amplifiers are defined as molecules that can covalently capture undetectable analytes after enzymatic signal generation and drag them into the pore for transduction. Here, we introduce converters as molecules that can shift the reactivity of amplifiers in situ to capture chemoorthogonal analytes. For this purpose, a series of dialkoxynaphthalene (DAN) and dialkoxyanthracene (DAA) hydrazinoboronic acids was prepared in situ from DAN and DAA hydrazides and formylphenylboronic acids. These converted amplifiers efficiently inactivate synthetic pores with internal naphthalenediimide clamps. This pore inactivation by DAN and DAA hydrazinoboronic acids vanishes in the presence of catechols such as (+)-catechin, presumably because the obtained boronate esters are too large to bind within the synthetic beta-barrel pore or because they prefer to partition into the bilayer membrane. The resulting increase in pore activity with increasing catechol concentration at constant amplifier concentration is shown to be compatible with the sensing of polyphenols in green tea.

Making Dimethylamino a Transformable Directing Group by Nickel-Catalyzed CN Borylation

The dimethylamino (Me2N) group is arguably the most versatile functional group capable of highly efficient and site-selective directed aromatic functionalizations at the ortho-, meta-, and para-positions depending on reaction conditions. While the repertoire of Me2N-directed reactions is growing at a rapid pace, the lack of a general method to transform this group to other functionalities hampers its wider application in organic synthesis. Here we report nickel-catalyzed C-N borylations of aryl- and benzyl-dimethylamines that permit the conversion of a huge library of largely underutilized Me2N-containing organic molecules into various functional molecules by taking advantage of the wealth of existing C-B functionalization methods.

Screening of π-Basic Naphthalene and Anthracene Amplifiers for π-Acidic Synthetic Pore Sensors

Synthetic ion channels and pores attract current attention as multicomponent sensors in complex matrixes. This application requires the availability of reactive signal amplifiers that covalently capture analytes and drag them into the pore. pi-Basic 1,5-dialkoxynaphthalenes (1,5-DAN) are attractive amplifiers because aromatic electron donor-acceptor (AEDA) interactions account for their recognition within pi-acidic naphthalenediimide (NDI) rich synthetic pores. Focusing on amplifier design, we report here the synthesis of a complete collection of DAN and dialkoxyanthracene amplifiers, determine their oxidation potentials by cyclic voltammetry, and calculate their quadrupole moments. Blockage experiments reveal that subtle structural changes in regioisomeric DAN amplifiers can be registered within NDI pores. Frontier orbital overlap in AEDA complexes, oxidation potentials, and, to a lesser extent, quadrupole moments are shown to contribute to isomer recognition by synthetic pores. Particularly important with regard to practical applications of synthetic pores as multianalyte sensors, we further demonstrate that application of the lessons learned with DAN regioisomers to the expansion to dialkoxyanthracenes provides access to privileged amplifiers with submicromolar activity.

Design and Synthesis of Oligosaccharides that Interfere with Glycoprotein Quality‐control systems

Calnexin (CNX) and its soluble homologue calreticulin (CRT) are lectin‐like molecular chaperones that help newly synthesized glycoproteins to fold correctly in the rough endoplasmic reticulum (ER). To investigate the mechanism of glycoprotein‐quality control, we have synthesized structurally defined high‐mannose‐type oligosaccharides related to this system. This paper describes the synthesis of the non‐natural undecasaccharide 2 and heptasaccharide 16, designed as potential inhibitors of the ER quality‐control system. Each possesses the key tetrasaccharide element (Glc1Man3) critical for the CNX/CRT binding, while lacking the pentamannosyl branch required for glucosidase II recognition. These oligosaccharides were evaluated for their ability to bind CRT by isothermal titration calorimetry (ITC). As expected, each of them had a significant affinity towards CRT. In addition, these compounds were shown to be resistant to glucosidase II digestion. Their activities in blocking the chaperone function of CRT were next measured by using malate dehydrogenase (MDH) as a substrate. Their inhibitory effects were shown to correlate well with their CRT‐binding affinities, both being critically dependent upon the presence of the terminal glucose (Glc) residue.

Synthesis of fluorine substituted oligosaccharide analogues of monoglucosylated glycan chain, a proposed ligand of lectin-chaperone calreticulin and calnexin

As a part of a exploring the N-glycan-mediated glycoprotein quality control in endoplasmic reticulum, 2-fluorinated derivative Glcα1 → 3Man(F) 1, Glcα1 → 3Man(F)α1 → 2Man2, and Glcα1 → 3Man(F)α1 → 2Manα1 → 2Man 3 were synthesized in a concise manner. These oligosaccharides were subjected to binding studies with calreticulin by using isothermal titration calorimetry. It was revealed that disaccharide 1 was a poor ligand, while tri- (2) and tetrasaccharide (3) had observable affinity. Published in 2003.

Hydrazinoanthrylboronic acids as exciton‐coupled circular dichroism (ECCD) probes for multivalent catechols, particularly epigallocatechin gallate

We report the use of exciton-coupled circular dichroism (ECCD) spectroscopy in multianalyte sensing systems in complex matrices. To prepare ECCD sensors, anionic anthrylhydazides are reacted with formylphenylboronic acids to give hydrazinoanthrylboronic acids that in turn are reacted with multivalent catechols in aqueous solution. The ECCD signal between the anthracene chromophores in the resulting boronate ester products depends strongly on the structure of the boronic acid sensor and the polyphenol analyte. This dependence of the ECCD signal on analyte structure is interesting for sensing applications. Best ECCD response is found for epigallocatechin gallate (EGCG), a key polyphenol in green tea. Weakly bell-shaped pH profiles, sensitivity to ionic strength and decreasing ECCD with decreasing solvent polarity imply that the CD active product is stabilized by hydrophobic interactions between the anthracene chromophores and by formation of the conjugate bases of the boronic esters. Analyte screening reveals selectivity for divalent catechols, with effective concentrations down to EC(50) = 5 microM for EGCG. Monovalent or achiral catechols such as (+)-catechin, protocatechuate or homoprotocatechuate are not detected. However, the latter two become detectable when attached to a chiral, divalent 1,2-cyclohexylamine scaffold. Application of this simple, user-friendly ECCD system to polyphenol sensing in various green tea extracts delivers easily accessible and reproducible values in the expected range.

Chemical hijacking of auxin signaling with an engineered auxin–TIR1 pair

The phytohormone auxin, indole-3-acetic acid (IAA), regulates nearly all aspects of plant growth and development. Despite substantial progress in our understanding of auxin biology, delineating specific auxin response remains as a major challenge. Auxin regulates transcriptional response via its receptors, TIR1/AFB F-box proteins. Here we report an engineered, orthogonal auxin-TIR1 receptor pair, developed through a bump-and-hole strategy, that triggers auxin signaling without interfering with endogenous auxin or TIR1/AFBs. A synthetic, convex IAA (cvxIAA) hijacked the downstream auxin signaling in vivo both at the transcriptomic level and in specific developmental contexts, only in the presence of a complementary, concave TIR1 (ccvTIR1) receptor. Harnessing the cvxIAA-ccvTIR1 system, we provide conclusive evidence for the role of TIR1-mediated pathway in auxin-induced seedling acid growth. The cvxIAA-ccvTIR1 system serves as a powerful tool for solving outstanding questions in auxin biology and for precise manipulation of auxin-mediated processes as a controllable switch.

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.

Systematic synthesis and inhibitory activity of haloacetamidyl oligosaccharide derivatives toward cytoplasmic peptide:N-glycanase

A series of glycosyl haloacetamides were synthesized as potential inhibitors of cytoplasmic peptide:N-glycanase (PNGase), an enzyme that removes N-glycans from misfolded glycoproteins. Chloro-, bromo-, and iodoacetamidyl chitobiose and chitotetraose derivatives exhibited a significant inhibitory activity. No inhibitory activity was observed with of fluoroacetamididyl derivatives. Moreover, N-acetylglucosamine derivatives, β-chloropropionamidyl chitobiose, and chloroacetamidyl cellooligosaccharide derivatives did not show any activity. These results underscore the importance of the N-acetyl groups of chitobiose for PNGase recognition. In addition, reactivity and position of the leaving group at the reducing end are also important factors.