Yuta Murai

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.

Comprehensive synthesis of photoreactive (3-trifluoromethyl)diazirinyl indole derivatives from 5- and 6- trifluoroacetylindoles for photoaffinity labeling.

5- and 6-trifluoromethyldiazirinyl indoles were synthesized from corresponding bromoindole derivatives for the first time. They acted as mother skeletons for the comprehensive synthesis of various bioactive indole metabolites. These can be used in biological functional analysis as diazirine-based photoaffinity labels.

Hydrogen/deuterium exchange of cross-linkable α-amino acid derivatives in deuterated triflic acid

In this paper we report here a hydrogen/deuterium exchange (H/D exchange) of cross-linkable α-amino acid derivatives with deuterated trifluoromethanesulfonic acid (TfOD). H/D exchange with TfOD was easily applied to o-catechol containing phenylalanine (DOPA) within an hour. A partial H/D exchange was observed for trifluoromethyldiazirinyl (TFMD) phenylalanine derivatives. N-Acetyl-protected natural aromatic α-amino acids (Tyr and Trp) were more effective in H/D exchange than unprotected ones. The N-acetylated TFMD phenylalanine derivative afforded slightly higher H/D exchange than unprotected derivatives. An effective post-deuteration method for cross-linkable α-amino acid derivatives will be useful for the analysis of biological functions of bioactive peptides and proteins by mass spectrometry. Graphical Abstract Hydrogen-deuterium exchange of cross-linkable α-amino acid derivatives in deuterated triflic acid proceeded smoothly at low temperature.

Effective Synthesis of Optically Active Trifluoromethyldiazirinyl Homophenylalanine and Aroylalanine Derivatives with the Friedel-Crafts Reaction in Triflic Acid

The Friedel-Crafts reaction with 3-(3-methoxyphenyl)-3-(trifluoromethyl)-3H-diazirine and optically active N-TFA-Asp(Cl)-OMe in triflic acid afforded homophenylalanine derivatives without any loss of the optical purity.

Ralstonins A and B, Lipopeptides with Chlamydospore-Inducing and Phytotoxic Activities from the Plant Pathogen Ralstonia solanacearum

Ralstonia solanacearum has an orphan hybrid polyketide synthase-nonribosomal peptide synthetase gene cluster. We herein isolate its products (named ralstonins A and B) from R. solanacearum and elucidate their structures and biological activities. Ralstonins are unusual lipodepsipeptides composed of 11 amino acids (containing unique amino acids such as β-hydroxytyrosine and dehydroalanine) and a 3-amino-2-hydroxyoctadecanoic acid, and their production is controlled by quorum sensing, a mechanism of bacterial cell-cell communication. Ralstonins exhibited chlamydospore-inducing activity and phytotoxicity.

Effective Synthesis of Optically Active 3-Phenyl-3-(3-trifluoromethyl)diazirinyl Bishomophenylalanine Derivatives

- Effective incorporation of phenyldiazirine moiety on the acyl residue of L - and D - glutamic acid by Friedel-Crafts reactions with triflic acid developed simple preparation of bishomophenylalanine (bhPhe) for aromatics, which added a versatile and a reliable access to photoreactive peptide probes.

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.

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

Indole-3-Acetic Acid Produced by Burkholderia heleia Acts as a Phenylacetic Acid Antagonist to Disrupt Tropolone Biosynthesis in Burkholderia plantarii

Burkholderia heleia PAK1-2 is a potent biocontrol agent isolated from rice rhizosphere, as it prevents bacterial rice seedling blight disease caused by Burkholderia plantarii. Here, we isolated a non-antibacterial metabolite from the culture fluid of B. heleia PAK1-2 that was able to suppress B. plantarii virulence and subsequently identified as indole-3-acetic acid (IAA). IAA suppressed the production of tropolone in B. plantarii in a dose-dependent manner without any antibacterial and quorum quenching activity, suggesting that IAA inhibited steps of tropolone biosynthesis. Consistent with this, supplementing cultures of B. plantarii with either L-[ring-2H5]phenylalanine or [ring-2H2~5]phenylacetic acid revealed that phenylacetic acid (PAA), which is the dominant metabolite during the early growth stage, is a direct precursor of tropolone. Exposure of B. plantarii to IAA suppressed production of both PAA and tropolone. These data particularly showed that IAA produced by B. heleia PAK1-2 disrupts tropolone production during bioconversion of PAA to tropolone via the ring-rearrangement on the phenyl group of the precursor to attenuate the virulence of B. plantarii. B. heleia PAK1-2 is thus likely a microbial community coordinating bacterium in rhizosphere ecosystems, which never eliminates phytopathogens but only represses production of phytotoxins or bacteriocidal substances.

Distinct Metabolites for Photoreactive l-Phenylalanine Derivatives in Klebsiella sp. CK6 Isolated from Rhizosphere of a Wild Dipterocarp Sapling

Photoaffinity labeling is a reliable analytical method for biological functional analysis. Three major photophores—aryl azide, benzophenone and trifluoromethyldiazirine—are utilized in analysis. Photophore-bearing l-phenylalanine derivatives, which are used for biological functional analysis, were inoculated into a Klebsiella sp. isolated from the rhizosphere of a wild dipterocarp sapling in Central Kalimantan, Indonesia, under nitrogen-limiting conditions. The proportions of metabolites were quite distinct for each photophore. These results indicated that photophores affected substrate recognition in rhizobacterial metabolic pathways, and differential photoaffinity labeling could be achieved using different photophore-containing l-phenylalanine derivatives.