Bis-Argentivorous Molecules Bridged by Phenyl and 4,4'-Biphenyl Groups: Structural and Dynamic Behavior of Silver Complexes.

Bis-argentivorous molecules (La and Lb), which have phenyl and 4,4'-biphenyl groups as linkers, have been prepared. The structures of Ag+ complexes with the new ligands (La and Lb) were investigated in solution and the solid state. The CSI-MS and 1H NMR titration of La and Lb with Ag+ show 1:1 and 1:2 complexes depending on the [Ag+]:[L] ratios. In the solid-state structures, single crystals of La and Lb with 2 equiv of Ag+ were prepared. X-ray crystallography of the silver(I) complexes with La and Lb showed that an intramolecular racemic structure (Δ(δδδδ)Λ(λλλλ) form) and a racemic mixture of Δ(δδδδ)Δ(δδδδ) and Λ(λλλλ)Λ(λλλλ) forms were formed, respectively. The dynamic 1H NMR studies suggest the following: (i) the activation entropies (ΔS⧧) of the side arm rotations in the Ag+ complex with La were all negative, indicating restricted rotation of the side arms due to their shortness, and (ii) the ΔS⧧ values of the Ag+ complexes with Lb were negative only when the side arms of both cyclens rotated simultaneously, and the ΔS⧧ values for the 1:1 and 1:2 complexes were positive when one cyclen side arm was rotated. These values of ΔS⧧ indicate that the biphenyl side arms between the two cyclens are not long enough to rotate the ring freely.

Cosmosen: Octa-Armed 24-Membered Cyclic Octaamine Synthesized from a Byproduct in the Preparation of 4-Benzyl-2,6-dioxocyclen.

The synthesis of an octa-armed 24-membered cyclic octaamine (1) is reported. When 4-benzyl-1,4,7,10-tetraazacyclododecane-2,6-dione (3a) was prepared by the reaction of diethylenetriamine with diethyl N-benzyliminodiacetate (2), a dimeric macrocycle (3b) was obtained as a byproduct in a 5% yield. An octa-armed 24-membered cyclic octaamine (1), named Cosmosen, was prepared via the reductive amination and reduction of 3b. The binding constants for the 1:1 and 2:1 (Ag+/1) complexation of 1 were estimated to be ca. 7.9 and 13.9, respectively, by titration experiments using UV-vis spectrometry in methanol and chloroform (v/v, 9:1) solutions at 298 K.

Inclusion of alkyl nitriles by tetra-armed cyclens with styrylmethyl groups.

Synthesis of tetra-armed cyclens (2a-2e), with substituted styrylmethyl groups as side-arms, and their Ag+ complexes is reported. The Ag+ complex with a tetra styrylmethyl-armed cyclen (2a) incorporates alkyl nitriles in a pseudo-cavity formed by the four styrylmethyl side-arms. This prompted us to apply this system to the determination of the absolute configurations of chiral nitriles with low [α]D and low circular dichroism (CD) intensity. In the CD spectra of the 2a/Ag+ complex, (S)- and (R)-G1 did not show a specific Cotton effect, while when chiral nitriles were added to the 2a/Ag+ complex, drastic spectral changes were observed. The (S)-G1@2a/Ag+ system exhibited first a negative and then a positive Cotton effect, whereas the (R)-G1@2a/Ag+ system showed the mirror image of the Cotton effect of (S)-G1@2a/Ag+. We have, therefore, demonstrated a new technique for determining the absolute configurations of weak optical rotation molecules using the Ag+ complex with 2a.

C-HCl- hydrogen bonds in solution and in the solid-state: HgCl2 complexes with cyclen-based cryptands.

Structural evidence is reported for C-HCl- hydrogen bonds in solution and in the solid state of HgCl2 complexes with cyclen-based cryptands. These cyclen-based cryptands (1) and (2) are bridged by di- and triethylene glycol units, respectively, between two aromatic rings. The X-ray structure indicates that the 2/HgCl2 complex contains an acetonitrile molecule in the cavity.

Identification of Cereulide-Producing Bacillus cereus by Nucleic Acid Chromatography and Reverse Transcription Real-Time PCR.

RNA extracts were analyzed with a nucleic acid sequence-based amplification (NASBA) - nucleic acid chromatography and a reverse transcription-quantitative PCR assay (RT-qPCR) based on the TaqMan probe for identification of cereulide-producing Bacillus cereus. All 100 emetic B. cereus strains were found to give positive results, but 50 diarrheal B. cereus strains and other bacterial species showed negative results in the NASBA-chromatography. That is, the assay could selectively identify the emetic strains among B. cereus strains. Also, the B. cereus contents of more than 10(7) cfu/ml were required for the identification of the cereulide-producing strains in this assay. In qRT-PCR assays, all 100 emetic type strains of B. cereus produced 10(2) - 10(4) copy numbers per ng of the RNA preparation, and the strains produced 10(4) copies including ones which had the high vacuolation activities of HEp-2 cells.

Evaluation of the immunochromatographic device for the detection of verotoxins in cultures of food materials.

The immunochromatographic assay, which targets Shiga toxin 1/verotoxin 1 (VT1) and/or Shiga toxin 2/verotoxin 2 (VT2) independently with same test device, was used for easily, rapidly and specifically detecting verotoxin-producing Escherichia coli among E. coli strains from food and fecal materials. All 10 strains of VT 1 and/or VT 2- producing E. coli among E. coli isolates from various sources showed a positive reaction to VT1- or VT2- antibodies, but other gram-negative and positive bacterial species had a negative reaction. Bacterial counts of 10(8) cfu/ml in enrichment broth and food suspension were required for the detection of VT-producing E. coli. The IC assay described here could detect easily and specifically the verotoxin-producing E. coli within 20 min by pure culture.

Detection of emetic Bacillus cereus by real-time PCR in foods.

The simplex real-time PCR assays based on the TaqMan probe and SYBR green I， which target cereulide synthetase genes (ces genes) were used for rapidly, reliably and sensitively identifying the emetic strains from among Bacillus cereus strains isolated from different sources. Only the emetic strains showed positive reactions to the real-time PCR assays, but all examined strains of diarrheal B. cereus, other Bacillus species and other gram positive and negative bacteria gave negative results. The final identification of emetic B. cereus was possible within 1 to 1.5 h in both simplex real-time PCR procedures. The detection limit of emetic strains in food was suggested to be 10(4)-10(5) cfu/g. Both simplex real-time PCR assays were found to be rapid, sensitive and reliable diagnostic tools that complement the different cellular, immunological and chemical detection methods for cereulide- producing B. cereus, even if a relatively expensive device is required for the assays.

Evaluation of immunochromatography for the rapid and specific identification of Listeria monocytogenes from food.

To rapidly, simply and specifically detect and identify Listeria monocytogenes from food samples, an immunochromatographic assay, based on gold-labeled monoclonal antibodies directed against an antigen common to all serovars of L. monocytogenes, was used. All strains of L. monocytogenes serovars showed a positive reaction to the assay, but all other gram positive and negative bacteria did not. The detection limit of the assay was in the order of 10(6) cfu/ml in fluid medium. The assay could simply and rapidly identify L. monocytogenes within 30 min by a pure culture without special instruments. Even if the selective enrichment cultivation was employed for the isolation and growth of bacteria from food materials, the application of the assay system could detect and identify L. monocytogenes precisely in various food materials within 2 to 3 days.

LC-MS analysis of the emetic toxin, cereulide, produced by Bacillus cereus.

A quantitative and chemical assay of cereulide produced in the cultures by some strains of Bacillus cereus was performed on a HPLC and a ESI electrospray ion trap mass analyzer, using the synthetic cereulide as a standard. All 20 strains of emetic B. cereus were found to produce 27 - 740 ng/ml of cereulide by the LC-MS analysis. In contrast, none of the 10 diarrheal strains produced it. 10(2) cfu/ml of the cereulide producible strain with a 210 ng/ml yield was inoculated into the 10% suspensions of 14 food products, and was incubated at 32°C for 24h. The B. cereus counts in the cultures grew in the order of 10(8) to 10(9) cfu/ml, although the bacteria could not grow in fruits, and the yields of cereulide ranged from 5.18µg in curry to 0.03µg/g of raw material and/or powder material, except for fruits. These culture supernatants were also tested for the biological activity in the HEp-2 cell culture assay. Consequently, a certain correlation was shown between the yields of cereulide and the HEp-2 vacuolation activities. In addition, the supernatants were administered i.p. to 5 Suncus marinus test animals. The emetic dose was calculated to be approximately 16µg/kg.