Makoto Hashimoto

A novel biotinylated heterobifunctional cross-linking reagent bearing an aromatic diazirine.

The synthesis of a p-[(3-trifluoromethyl)diazirine-3-yl]benzoic acid derivative is described as a new carbene generating heterobifunctional cross-linking reagent. The cross-linker carries a biotin moiety in order to make use of avidin-biotin technology for specific manipulation of cross-linked components. To evaluate the ability of this reagent, the inter-subunit cross-linking of egg-white avidin tetramer was investigated. As a typical application of avidin-biotin technology for cross-linking experiments, a chemiluminescent detection method was examined to identify photobiotinylated components. A cross-linked dimeric product with an apparent molecular mass of 38 kDa was clearly visualized by the combined use of a horseradish peroxidase-streptavidin conjugate and a luminol-based chemiluminescent system.

Alternative One-Pot Synthesis of (Trifluoromethyl)phenyldiazirines from Tosyloxime Derivatives: Application for New Synthesis of Optically Pure Diazirinylphenylalanines for Photoaffinity Labeling

Alternative one-pot synthesis of 3-(trifluoromethyl)-3-phenyldiazirine derivatives from corresponding tosyloximes is developed. The deprotonation of intermediate diaziridine by NH2(-) is a new approach for construction of diazirine. Moreover, a novel synthesis of optically pure (trifluoromethyl)diazirinylphenylalanine derivatives was attempted involving these methods.

Beetroot betalain inhibits peroxynitrite-mediated tyrosine nitration and DNA strand cleavage

Two major betalains, red-purple betacyanins and yellow betaxanthins, were isolated from red beetroots (Beta vulgaris L.), and their peroxynitrite (ONOO − ) scavenging capacity was investigated. Apparent colours of the betalains were bleached by the addition of ONOO − , and the absorbance decreases were suppressed in the presence of glutathione, a ONOO − scavenger. After bleaching, a new absorption maximum was observed at 350 nm in the spectrum of the resulting reaction mixture. New peaks were detected from HPLC analysis of the reaction products of betanin, a representative constituent of red beetroot betacyanins, treated with ONOO − monitoring at 350 nm, and the intensity of the major peak was positively correlated with ONOO − concentration. Betanin inhibited the ONOO − (0.5 mM)-dependent nitration of tyrosine (0.1 mM). Additionally, the IC50 value of betanin (19.2 μM) was lower than that of ascorbate (79.6 μM). The presence of betanin (0.05–1.0 mM) also inhibited ONOO − (0.5 mM)-dependent DNA strand cleavage in a concentration-dependent manner. These results suggest that betalains can protect cells from nitrosative stress in addition to protecting them from oxidative stresses.

Sialyltransferases of marine bacteria efficiently utilize glycosphingolipid substrates

Bacterial sialyltransferases (STs) from marine sources were characterized using glycosphingolipids (GSLs). Bacterial STs were found to be beta-galacotoside STs. There were two types of STs: (1) ST obtained from strains such as ishi-224, 05JTC1 (#1), ishi-467, 05JTD2 (#2), and faj-16, 05JTE1 (#3), which form alpha2-3 sialic acid (Sia) linkages, named alpha2-3ST, (2) ST obtained from strains such as ISH-224, N1C0 (#4), pda-rec, 05JTB2 (#5), and pda-0160, 05JTA2 (#6), which form alpha2-6 Sia linkages, named alpha2-6ST. All STs showed affinity to neolacto- and lacto-series GSLs, particularly in neolactotetraosyl ceramide (nLc(4)Cer). No large differences were observed in the pH and temperature profiles of enzyme activities. Kinetic parameters obtained by Lineweaver-Burk plot analysis showed that #3 and #4 STs had practical synthetic activity and thus it became easily possible to achieve large-scale ganglioside synthesis (100-300 muM) using these recombinant enzymes. Gangliosides synthesized from nLc(4)Cer by alpha2-3 and alpha2-6STs were structurally characterized by several analytical and immunological methods, and they were identified as IV(3)alphaNeuAc-nLc(4)Cer(S2-3PG) and IV(6)alphaNeuAc-nLc(4)Cer (S2-6PG), respectively. Further characterization of these STs using lactotetraosylceramide (Lc(4)Cer), neolactohexaosylceramide (i antigen), and IV(6)kladoLc(8)Cer (I antigen) showed the synthesis of corresponding gangliosides as well. Synthesized gangliosides showed binding activity to the influenza A virus [A/panama/2007/99 (H3N2)] at a similar level to purified S2-3PG and S2-6PG from mammalian sources. The above evidence suggests that these STs have unique features, including substrate specificities restricted to lacto- and neolactoseries GSLs, as well as catalytic potentials for ganglioside synthesis. This demonstrates that efficient in vitro ganglioside synthesis could be a valuable tool for selectively synthesizing Sias modifications, thereby permitting the exploration of unknown functions.

Photoactive ligands probing the sweet taste receptor. Design and synthesis of highly potent diazirinyl D-phenylalanine derivatives

Some D-amino acids such as d-tryptophan and D-phenylalanine are well known as naturally-occurring sweeteners. Photoreactive D-phenylalanine derivatives containing trifluoromethyldiazirinyl moiety at 3- or 4-position of phenylalanine, were designed as sweeteners for functional analysis with photoaffinity labeling. The trifluoromethyldiazirinyl D-phenylalanine derivatives were prepared effectively with chemo-enzymatic methods using L-amino acid oxidase and were found to have potent activity toward the human sweet taste receptor.

Carot-4-en-9,10-diol, a conidiation-inducing sesquiterpene diol produced by Trichoderma virens PS1-7 upon exposure to chemical stress from highly active iron chelators.

ABSTRACT To screen biocontrol agents against Burkholderia plantarii, the causative agent of rice seedling blight, we employed catechol, an analog of the virulence factor tropolone, to obtain chemical stress-resistant microorganisms. The fungal isolate PS1-7, identified as a strain of Trichoderma virens, showed the highest resistance to catechol (20 mM) and exhibited efficacy as a biocontrol agent for rice seedling blight. During investigation of metabolic traits of T. virens PS1-7 exposed to catechol, we found a secondary metabolite that was released extracellularly and uniquely accumulated in the culture. The compound induced by chemical stress due to catechol was subsequently isolated and identified as a sesquiterpene diol, carot-4-en-9,10-diol, based on spectroscopic analyses. T. virens PS1-7 produced carot-4-en-9,10-diol as a metabolic response to tropolone at concentrations from 0.05 to 0.2 mM, and the response was enhanced in a dose-dependent manner, similar to its response to catechol at concentrations from 0.1 to 1 mM. Some iron chelators, such as pyrogallol, gallic acid, salicylic acid, and citric acid, at 0.5 mM also showed activation of T. virens PS1-7 production of carot-4-en-9,10-diol. This sesquiterpene diol, formed in response to chemical stress, promoted conidiation of T. virens PS1-7, suggesting that it is involved in an autoregulatory signaling system. In a bioassay of the metabolic and morphological responses of T. virens PS1-7, conidiation in hyphae grown on potato dextrose agar (PDA) plates was either promoted or induced by carot-4-en-9,10-diol. Carot-4-en-9,10-diol can thus be regarded as an autoregulatory signal in T. virens, and our findings demonstrate that intrinsic intracellular signaling regulates conidiation of T. virens.

Repression of Tropolone Production and Induction of a Burkholderia plantarii Pseudo-Biofilm by Carot-4-en-9,10-diol, a Cell-to-Cell Signaling Disrupter Produced by Trichoderma virens

Background The tropolone-tolerant Trichoderma virens PS1-7 is a biocontrol agent against Burkholderia plantarii, causative of rice seedling blight. When exposed to catechol, this fungus dose-dependently produced carot-4-en-9,10-diol, a sesquiterpene-type autoregulatory signal molecule that promotes self-conidiation of T. virens PS1-7 mycelia. It was, however, uncertain why T. virens PS1-7 attenuates the symptom development of the rice seedlings infested with B. plantarii. Methodology/Principal Findings To reveal the antagonism by T. virens PS1-7 against B. plantarii leading to repression of tropolone production in a coculture system, bioassay-guided screening for active compounds from a 3-d culture of T. virens PS1-7 was conducted. As a result, carot-4-en-9,10-diol was identified and found to repress tropolone production of B. plantarii from 10 to 200 µM in a dose-dependent manner as well as attenuate virulence of B. plantarii on rice seedlings. Quantitative RT-PCR analysis revealed that transcriptional suppression of N-acyl-L-homoserine lactone synthase plaI in B. plantarii was the main mode of action by which carot-4-en-9,10-diol mediated the quorum quenching responsible for repression of tropolone production. In addition, the unique response of B. plantarii to carot-4-en-9,10-diol in the biofilm formed in the static culture system was also found. Although the initial stage of B. plantarii biofilm formation was induced by both tropolone and carot-4-en-9,10-diol, it was induced in different states. Moreover, the B. plantarii biofilm that was induced by carot-4-en-9,10-diol at the late stage showed defects not only in matrix structure but also cell viability. Conclusions/Significance Our findings demonstrate that carot-4-en-9,10-diol released by T. virens PS1-7 acts as an interkingdom cell-to-cell signaling molecule against B. plantarii to repress tropolone production and induces pseudo-biofilm to the cells. This observation also led to another discovery that tropolone is an autoregulatory cell-to-cell signaling molecule of B. plantarii that induces a functional biofilm other than a simple B. plantarii virulence factor.

Novel Synthesis of Optically Active Bishomotyrosine Derivatives Using the Friedel-Crafts Reaction in Triflic Acid

We report here a novel synthesis of optically active bishomotyrosine. The bishomotyrosine skeleton was constructed by using a Friedel-Crafts reaction between phenol and optically active N-Tfa-Glu(Cl)-OMe in triflic acid under the mild condition. Reduction and subsequent deprotection then afforded bishomotyrosine derivatives without any loss of optical purity.

Dehydrogenation of the NH−NH Bond Triggered by Potassium tert‐Butoxide in Liquid Ammonia

A novel strategy for the dehydrogenation of the NH-NH bond is disclosed using potassium tert-butoxide (tBuOK) in liquid ammonia (NH3 ) under air at room temperature. Its synthetic value is well demonstrated by the highly efficient synthesis of aromatic azo compounds (up to 100u2009% yield, 3u2005min), heterocyclic azo compounds, and dehydrazination of phenylhydrazine. The broad application of this strategy and its benefit to chemical biology is proved by a novel, convenient, one-pot synthesis of aliphatic diazirines, which are important photoreactive agents for photoaffinity labeling.

Simple and Stereocontrolled Preparation of Benzoylated Phenylalanine Using Friedel–Crafts Reaction in Trifluoromethanesulfonic Acid for Photoaffinity Labeling

Simple and stereocontrolled preparation of benzoylated phenylalanine derivatives from optically pure phenylalanine using Friedel‒Crafts reaction in trifluoromethanesulfonic acid (TfOH) is reported; these derivatives are useful for photoaffinity labeling. Protected or unprotected phenylalanine derivatives were converted to benzoyl derivatives in TfOH at room temperature in a short time without loss of optical purity. The reaction condition was applied to synthesize novel photoreactive phenylalanine derivative, which has two photophores (benzophenone and diazirine). The detail analysis of photo-irradiation for two different photophores contained phenylalanine derivative was also investigated. Photoaffinity labeling is a valuable chemical method for studying the interactions of biologically active molecules with their target proteins. In photoaffinity labeling, a covalent bond is formed between ligand and target proteins upon irradiation with UV light. For instance, to study biologically active peptide interactions for a target protein, a photoreactive α-amino acid will be required and is a powerful probe for photoaffinity labeling. It has been reported for the preparation of benzophenone contained α-amino acid derivatives, which were synthesized from halo methyl derivatives of benzophenone and α-amino malonate equivalents using racemic or asymmetric methods (Figure 1A). But there are no reports on direct construction of benzophenone moiety on phenylalanine with Friedel‒Crafts reaction due to low solubility of phenylalanine derivatives in appropriate solvent for the reaction. Here, we report a useful strategy for direct construction of benzophenone moiety on optically pure phenylalanine using a Friedel‒ Crafts reaction in trifluoromethanesulfonic acid (TfOH) with stereocontrolled manner (Figure 1B). Figure 1. Synthesis of benzoylated phenylalanine derivatives (A) previous report, (B) this report. We have recently reported that TfOH can effectively dissolve α-amino acid derivatives and catalyze Friedel-Crafts reaction. We attempted direct construction of benzoylated phenylalanine by using TfOH (Scheme 1). Optically pure unprotected phenylalanine (L-1 or D-1) was dissolved in TfOH at 0 °C and stirred for 1 h, followed by the addition of benzoyl chloride (5, 8 eq). The reaction mixture was stirred at room temperature. Although the starting material was consumed completely in 12 h, the reaction became a complex mixture. The isolated benzoyl-phenylalanine (6) was less than 30 % yield without loss of optical purity (Table 1, Entries 1-2). No improvement was observed, when the reaction mixture was heated (Table 1, Entries 3-4). Next, we considered to prevent the acid-base reaction between amino group and TfOH. Optically pure N-Ac-L-Phe (L-2) was subjected to benzoylation at room temperature, 60 C and 80 C. Although chemical yield was improved using L-2, the product (7) was loss of its optical activity (Table 1, Entries 5-7). Optically pure N-Ac-L-Phe-OMe (L-3) was subject to Friedel-Crafts reaction at room temperature. Consumption of L-3 and construction of benzophenone were observed within 1 h. But the α-amino acid skeleton was converted to oxazole derivative (9) between acetamide and methyl ester (Table 1, Entry 8). And we observed that L-3 did not form an oxazole derivative 9 without benzoyl chloride 5 under the same condition. These results indicated that oxazole formation was promoted by in situ formation of mixed anhydride from benzoyl chloride and TfOH then the mixed anhydride reacted with 3. It is consistent with the formation of oxazoles from N-acyl amino acid methyl esters in the presence of triflic anhydride. To prevent oxazole formation, we changed the protecting group for amino moiety from acetamide to trifluoroacetamide: the trifluoromethyl group is highly electron-withdrawing and sterically-bulky to prevent imino triflate formation. N-TFA-L-Phe-OMe (L-4) was treated at room temperature and completely consumed for 10 h. The yield of desired product L-8 improved by using compound L-4 without loss of optical purity (Table 1, Entry 9). The same treatment of D-4 was also afforded D-8 without loss of optical purity (Table 1, Entry 10). Scheme 1. Simple and stereocontrolled preparation of benzoylated phenylalanine derivatives using Friedel–Crafts reaction in trifluoromethanesulfonic acid. Entry Material Condition Product Yield (%) ee (%) 1 L-1 rt, 12 h L-6 28 98 2 D-1 rt, 12 h D-6 29 98 3 L-1 60 °C, 1 h L-6 26 98 4 L-1 80 °C, 1 h L-6 18 98 5 L-2 rt, 48 h L-7 44 74 6 L-2 60 °C, 2 h L-7 58 79 7 L-2 80 °C, 1 h L-7 59 9 8 L-3 rt, 1 h 9 63 9 L-4 rt, 10 h L-8 45 98 10 D-4 rt, 10 h D-8 41 98 Table 1. The conditions and results of Friedel‒Crafts reaction; Determination of enantiomeric excess: a Chirobiotic T (Astec), condition; methanol/water = 1/9, b as N-Ac-Phe(benzoyl)-OMe after methylation with thionyl chloride in MeOH at room temperature for 24 h, CHIRALPAK AD (Daicel), condition; hexane/iso-propanol = 7/3, c Compound 8 converted to compound 6 with 1 M NaOH, condition the same of a. (Trifluoromethyl)phenyldiazirine (TPD) is one of the important photophores in photoaffinity labeling. Although TPD is expensive and sensitive to high temperature under strong acid condition, we have reported that TPD can be applied to the Friedel‒Crafts reaction in TfOH at room temperature to synthesize TPD contained (bis) homophenylalanine derivatives. Although both benzophenone and TPD are excited at 350 nm, the reactive species generated from TPD was faster than that of benzophenone. But it has not been reported two photophores were constructed in one molecule and comprehensive analysis of photoreactive properties. We tried to construct two kinds of photophore (benzophenone and diazirine) on phenylalanine using our development synthesis. N-TFA-L-Phe-OMe (L-4) was treated with benzoyl chloride derivative (11) in TfOH at room temperature until L-4 was consumed completely. The desired product (L-12) was obtained in high yield without decomposition of diazirine moiety. Deprotection of L-12 proceeded with 1 M NaOH to yield compound L-13 (Scheme 2). The same treatment of N-TFA-D-Phe-OMe (D-4) afforded optically active of D-13 as above without loss of optical purity. Scheme 2. Synthesis of compound (12) using Friedel‒Crafts reaction in TfOH. Determination of enantiomeric excess: Chirobiotic T (Astec), condition; methanol/water = 1/9. We also confirmed the photoreactive properties of 13. We have already demonstrated that the concentration of the diazirinyl compound had to be set to less than 1 mM to minimize isomerization to the diazo compound. Methanolic solution of L-13 was irradiated with black light (100 W) for 10 min. The maximum absorption at 350 nm for diazirine moiety of L-13 decreased with time-dependent manner (Figure 2A). The half-life of L-13 was determined to be 35 seconds. The photolyzed mixture was subjected to UPLC-TOF-MS. The major product afforded a molecular weight of m/z 382, which was consisted with methanol insertion to diazirine moiety and benzophenone is still remained (L-14a, Figure 2B, I). One of the minor product afforded a molecular weight of m/z 414, which arose from the reaction of both photophores with methanol (L-15a, Figure 2C, I). It was slightly difficult to predict detail structure of the reaction product in methanol from mass spectrometric analysis. Deuterated methanol (CD3OD) was subjected to photolysis. For major product, which was activated only TPD, 4 mass number increased, m/z 386 in CD3OD (Figure 2B, II). The result indicated “D” and “OCD3” were incorporated to the diazirine moiety (L-14b). On the other hand, minor product, which was activated both TPD and benzophenone, afforded 6 mass number increased peak m/z 420 (L-15b). A) B) major product C) minor product Figure 2. Photolysis of 1 mM of compound L-13 in methanol or deuterated methanol with 100 W black light in 10 minutes. A) UV-vis spectral change of photolyzed mixture. B) Mass spectrum of the major product photolyzed L-13 in methanol or deuterated methanol. C) Mass spectrum of the minor prouct photolyzed L-13 in methanol or deuterated methanol. These results indicated that benzophenone part increased two mass units. The photoirradiated structure was considered to be generated by the bond formation between carbonyl carbon of benzophenone and carbon of methanol (Figure 2C, II). Additionally we confirmed that methanolic or deuterated methanolic solution of benzophenone derivatives 6 did not decompose by photo-irradiation within 10 min. These results indicated that diazirine photophore substitutions at p-position of benzophenone promoted activation of benzophenone faster than unsubstituted one. In summary, Friedel‒Crafts reaction with TfOH as solvent and catalyst enabled us to employ simple and stereocontrolled benzoylation of optically active phenylalanine to prepare photoreactive phenylalanine derivatives. Additionally, this synthetic method was applicable to construction of two kinds of photophore (benzophenone and diazirine) in phenylalanine derivative. And photolysis study of 13 revealed that we could control the reactivity between TPD and benzophenone in the same molecule by photo-irradiation times. These simple preparations will be acceptable for all biochemist and contribute to understanding peptide-receptor interactions with photoaffinity labeling. EXPERIMENTAL H, C and F NMR spectra were measured by JEOL ECA 500 and EX 270 spectrometers for structural determinations. ESI-TOF-MS spectra were measured with a Waters LCT Premier XE spectrometer. General procedure for Friedel-Crafts reaction TfOH (0.5 mL) was added to phenylalanine derivatives (0.1 mmol) at 0 °C in a tube with screw cap and PTFE-faced rubber liner. After solution became homogeneous, benzoyl chloride (3~8 equivalents) was added at 0 °C. The reaction mixture was stirred at indicated temperature until the stating material was consumed completely, then poured into cold water and