A simple, rapid, and efficient method for generating multigene-knockout culture cells by the CRISPR/Cas9 system.

We evaluated the efficacy of simultaneous multiple-gene knockout in human culture cells. By simple co-transfection of HeLa cells with a mixture of pX330-based targeting plasmids together with a puromycin resistance plasmid, followed by transient selection of puromycin-resistant cells, Cas9/single-guide RNA (sgRNA)-transduced polyclonal cell populations were selected and grown. Western blot analyses revealed co-transfection of up to seven targeting plasmids for p38α, p38ß, JNK1, JNK2, Mnk1, ERK1, and mLST8 genes, drastically reduced protein expression of these genes in the polyclonal population. Analyses of a randomly isolated group of 25 clones revealed knockout efficiencies for the seven targeted genes ranging between 68% and 100%, and in six clones (24%), all targeted genes were disrupted. Deep sequencing analyses of the individual target sites revealed that, in most cases, Cas9/sgRNA-induced nonhomologous end joining resulted in deletion or insertion of only a few base pairs at the break points. These results demonstrate that simple co-transfection-based simultaneous targeting offers an easy, rapid, and efficient method to generate multiplex gene-knockout cell lines.

Intramolecular axial α/ß-coordination of the 132-terminal pyridyl group to the central zinc atom in chlorophyll-a derivatives.

Chlorophyll(Chl)-a derivatives possessing a zinc center and a C132-alkanoate residue with a terminal pyridyl group were synthesized. Their C132-epimerically pure products were isolated by preparative reverse phase HPLC. The C132-stereochemistry resulted in two directed terminal pyridinyl groups that coordinate with the central zinc atom in each stereoisomeric molecule: α/ß-intramolecular axial coordination. The asymmetric axial coordinations mimic the immobilization manner of Chl-a in photosynthetically active proteins. The diastereomerically dependent conformers in solution were characterized by 1D/2D NMR, UV-visible absorption, and fluorescence emission as well as circular dichroism (CD) spectroscopy. The 1H NMR and DOSY spectra revealed that the stereoselectively intramolecular coordination occurred in less coordinative deuterated chloroform, whereas a highly coordinative deuterated pyridine molecule replaced the terminal pyridine moiety as the axial ligand to form a mixture of α/ß-coordinated species. Their optical spectra in pyridine were nearly independent of the C132-stereochemistry and the linkers in the C132-substituents. The CD bands of ß-coordinated species in chloroform were more intense than those of the corresponding α-coordinated stereoisomers, indicating that the former had a larger distortion of the chlorin π-plane than the latter. Therefore, the α-coordinated Chl complex is more conformationally stable than the ß-complex.

Synthesis of chlorophyll-a homologs by C132-substitutions and their physico- and biochemical properties.

A mixture of pheophytins-a/a', metal-free forms of photosynthetically active chlorophyll(Chl)s-a/a' bearing the 132-methoxycarbonyl group, was substituted at the C132-position by bimolecular nucleophilic substitution with methyl bromoacetate or Michael addition with methyl acrylate, followed by C132-demethoxycarbonylation and magnesium insertion at the central position, to afford Chl-a/a' homologs possessing a methoxycarbonylmethyl or 2-methoxycarbonylethyl group at the C132-position, respectively. These C132-methylene- and ethylene-inserted homologs were characterized by 1D/2D 1H NMR spectroscopy, and the optical properties of their C132-epimerically pure samples are investigated using visible absorption, fluorescence emission, and circular dichroism spectroscopies. The stereochemistry at the C132-chiral center of these Chl-a/a' homologs was not inverted in a basic solution, and the Chl-a homologs were effective for the substrates for the chlorophyllase reaction, hydrolysis of the phytyl ester.

In vitro C132-dealkoxycarbonylations of zinc chlorophyll a derivatives including C132-substitutes by a BciC enzyme.

Chlorosomes in the green photosynthetic bacteria are the largest and most efficient light-harvesting antenna systems of all phototrophs. The core part of chlorosomes consists of bacteriochlorophyll c, d, e, or f molecules. In their biosynthetic pathway, a BciC enzyme catalyzes the removal of the C132-methoxycarbonyl group of chlorophyllide a. In this study, in vitro C132-dealkoxycarbonylations of zinc chlorophyll a derivatives bearing a methyl-, ethyl- or propyl-esterifying group and its methyl ester analogs with additional alkyl and hydroxy groups at the C132-position were examined using the BciC enzyme. The BciC-catalyzed reaction activity for the C132-methoxycarbonylated substrate was comparable to that for the ethoxycarbonylated compound; however, depropoxycarbonylation did not proceed. The BciC enzymatic demethoxycarbonylation of zinc methyl C132-alkylated pheophorbides a was gradually suppressed with the elongation of the alkyl chain and finally became inactive for the propyl substrate. The reaction of the C132-hydroxylated substrate (allomer) was accelerated compared to that of the C132-methyl analog possessing a similar steric size, and gave the corresponding C132-oxo product via further air-oxidation. All of the abovementioned enzymatic reactions occurred for one of the C132-epimers with the same configuration as in chlorophyllide a. The above substrate specificities and product distributions indicated the stereochemistry and size of the BciC enzymatic active site (pocket).

Visible-light driven hydrogen production using chlorophyll derivatives conjugated with a viologen moiety in the presence of platinum nanoparticles.

The utilization of the light-harvesting and electron-transferring function of chlorophylls (Chls) has received attention for visible-light driven hydrogen production. In this work, a series of Chl derivatives based on pyropheophorbide-a (Pyro-a) conjugated with a viologen moiety, including a Pyro-a methyl ester directly bonded with the viologen at the 3-position 1, its 31-methylene analog 2 and Pyro-a connected with the viologen in the 17-substituent 3, were synthesized from chemical modification of naturally occurring Chl-a and characterized in terms of their photochemical and photophysical properties. As the photoexcited singlet state of the Pyro-a moiety was strongly quenched by the viologen moiety in a molecule, the effective photoinduced intramolecular electron transfer from Pyro-a to the bonded viologen moiety occurred. Moreover, these molecules were applied as a photosensitizer in the system for visible-light driven hydrogen production with platinum nanoparticles via intramolecular reduction of the bonded viologen moiety. Efficient photoreduction of external methyl viologen and successive hydrogen production on platinum nanoparticles were achieved using the synthetic conjugate of Pyro-a with the viologen moiety as a photosensitizer. In particular, effective visible-light driven hydrogen production was accomplished using 3 and platinum nanoparticles via the reduction of external methyl viologen.

C31-Selective substitution of cationic N-heteroaromatic groups into a 3-vinylated chlorophyll-a derivative.

A variety of N-containing heteroarenes were site-selectively introduced at the C31-position of a chlorophyll-a derivative possessing a 3-vinyl group through a C-N+ bond via oxidative reactions using iodine in the presence of a silver(i) salt. Electron-rich N-heteroarenes were effectively substituted, while electron-withdrawing or bulky nitrogen-neighboring substituents suppressed the reactivities. The cationic products formed were characterized by 1D/2D NMR, and their optical properties were also investigated.

Nanotubes of Biomimetic Supramolecules Constructed by Synthetic Metal Chlorophyll Derivatives.

Various supramolecular nanotubes have recently been built up by lipids, peptides, and other organic molecules. Major light-harvesting (LH) antenna systems in a filamentous anoxygenic phototroph, Chloroflexus (Cfl.) aurantiacus, are called chlorosomes and contain photofunctional single-wall supramolecular nanotubes with approximately 5 nm in their diameter. Chlorosomal supramolecular nanotubes of Cfl. aurantiacus are constructed by a large amount of bacteriochlorophyll(BChl)-c molecules. Such a pigment self-assembles in a chlorosome without any assistance from the peptides, which is in sharp contrast to the other natural photosynthetic LH antennas. To mimic chlorosomal supramolecular nanotubes, synthetic models were prepared by the modification of naturally occurring chlorophyll(Chl)-a molecule. Metal complexes (magnesium, zinc, and cadmium) of the Chl derivative were synthesized as models of natural chlorosomal BChls. These metal Chl derivatives self-assembled in hydrophobic environments, and their supramolecules were analyzed by spectroscopic and microscopic techniques. Cryo-transmission electron microscopic images showed that the zinc and cadmium Chl derivatives could form single-wall supramolecular nanotubes and their outer and inner diameters were approximately 5 and 3 nm, respectively. Atomic force microscopic images suggested that the magnesium Chl derivative formed similar nanotubes to those of the corresponding zinc and cadmium complexes. Three chlorosomal single-wall supramolecular nanotubes of the metal Chl derivatives were prepared in the solid state and would be useful as photofunctional materials.

Asymmetric Diels-Alder Reaction of α-Substituted and ß,ß-Disubstituted α,ß-Enals via Diarylprolinol Silyl Ether for the Construction of All-Carbon Quaternary Stereocenters.

The asymmetric Diels-Alder reaction of α-substituted acrolein proceeds in the presence of the trifluoroacetic acid salt of trifluoromethyl-substituted diarylprolinol silyl ether to afford the exo-isomer with both excellent diastereoselectivity and high enantioselectivity. In the Diels-Alder reaction of a ß,ß-disubstituted α,ß-unsaturated aldehyde, good exo-selectivity and excellent enantioselectivity was obtained when the perchloric acid salt of the bulky triisopropyl silyl ether of trifluoromethyl substituted diarylprolinol was employed as an organocatalyst in the presence of water. In both cases, all-carbon quaternary stereocenters are constructed enantioselectively.

Synthesis of methyl (13(2)R/S)-alkyl-pyropheophorbide a and a non-epimerized chlorophyll a mimic.

The (13(2)R/S)-methoxycarbonyl group of methyl pheophorbides a/a' (chlorophyll a/a' derivatives) was converted to methyl, ethyl, propyl, and isopropyl groups through the C13(2)-alkylation under basic conditions followed by pyrolysis in 2,4,6-collidine with lithium iodide. All the resulting products, methyl 13(2)-alkyl-pyropheophorbides a, predominantly gave the (13(2)R)-stereoisomers with about one tenth of the (13(2)S)-epimers. Their stereochemistry was determined by 1D/2D NMR and their optical properties were characterized by visible absorption and circular dichroism spectroscopy. Methyl (13(2)R)-propyl-pyropheophorbide a was converted to (13(2)R)-propyl-pyrochlorophyll a by ester exchanging and magnesium chelating reactions. The synthetic chlorophyll a analogue showed non-epimerization at the 13(2)-position in pyridine-d5 at 40°C, while naturally occurring chlorophyll a was easily epimerized under the same conditions to give its epimeric mixture.

Extra-Annulated Chlorophyll Derivatives by Scholl Reaction.

The ferric chloride assisted Scholl reaction of methyl (3,5-dimethoxyphenyl)carbonyl-pyropheophorbide-a produced an extra-annulated, didehydrogenated chlorophyll derivative with a six-membered ring fused at the pyrrole A-ring. The steric interaction of the substituents on the additional tetralone and B-rings induced molecular helicity. The helically cyclized stereoisomers were separated by recrystallization, and their (P/M)-stereochemistry was confirmed by circular dichroism spectra. The distortion of their chlorin π-systems broadened electronic absorption bands to cover all visible regions and reach the near-infrared region.

Palladium nanoparticles captured in microporous polymers: a tailor-made catalyst for heterogeneous carbon cross-coupling reactions.

A new strategy based on polymerization-induced phase separation (PIPS) techniques was proposed for fabricating palladium nanoparticles (PdNPs) captured in a microporous network polymer. Pd(OAc)(2) was premixed with a monomer having a poly(amidoamine)-based dendrimer ligand, and subsequently this was thermally polymerized with an excess amount of ethylene glycol dimethacrylate under PIPS conditions. In this system, the formation of PdNPs occurred concurrently with the polymer synthesis in a one-pot process, even with no additional reducing reagent. The resultant microporous polymer was found to have a mesoporosity; the nitrogen sorption analysis gave a specific-surface area of 511 m(2) g(-1), an average pore diameter of 9.9 nm, and a total pore volume of 1.01 mL g(-1). The TEM images of the polymer revealed that the created PdNPs were very small with a diameter of mainly ca. 2.0 nm; the high-resolution images were lattice-resolvable, showing the crystalline nature of the PdNPs (Pd(111) facets). Catalytic performances of the PdNP-containing microporous polymers were investigated for a heterogeneous Suzuki-Miyaura reaction of 4'-bromoacetophenone and phenylboronic acid in water. In the presence of 10(-2) molar equiv of the polymer, the reaction efficiently proceeded at 80 degrees C and gave the desired product, 4-acetylbiphenyl, in >90% yield after 2 h. On the basis of the ICP-AES analysis, the Pd content released into the solution phase was estimated to be only 0.27% of the initial charge. Thereby, this polymer was successfully recovered by simple filtration and reused with only a minimal loss of activity (yield >90% even at the eighth run). When the catalytic reaction was examined with a low amount of the polymer catalyst, the turnover number (TON) reached 8.5 x 10(4) while maintaining a good yield. Finally, the dendrimer template effect of the polymer catalyst was discussed by referring to the catalytic performances of a control polymer prepared with nonintegrated ligand monomers.

Structural requirements of strigolactones for hyphal branching in AM fungi.

Strigolactones are a group of terpenoid lactones that act as a host-derived signal in the rhizosphere communication of plants with arbuscular mycorrhizal (AM) fungi and root parasitic weeds as well as an endogenous plant hormone regulating shoot branching in plants. Strigolactones induce hyphal branching in AM fungi at very low concentrations, suggesting a highly sensitive perception system for strigolactones present in AM fungi. However, little is known about the structural requirements of strigolactones for hyphal branching in AM fungi. Here, we tested a series of natural and synthetically modified strigolactones as well as non-strigolactone-type germination stimulants for hyphal branching-inducing activity in germinating spores of the AM fungus Gigaspora margarita. All tested compounds with a tricyclic lactone coupled to a methylbutenolide via an enol ether bond showed activity, but differed in the active concentration and in the branching pattern of hyphae. Truncation of the A- and AB-rings in the tricyclic ABC lactone of strigolactones resulted in a drastic reduction in hyphal branching activity. Although the connection of the C-ring in the tricyclic lactone to the methylbutenolide D-ring was shown to be essential for hyphal branching, the bridge structure in the C-D part was found not necessarily to be enol ether, being replaceable with either alkoxy or imino ethers. These structural requirements in AM fungi are very similar but not identical to those observed in root parasitic weeds, especially with respect to the enol ether bridge in the C-D part.

Enzymatic Preparation and Oxidative Stability of Human Milk Fat Substitute Containing Polyunsaturated Fatty Acid Located at sn-2 Position.

The development of human milk fat substitutes (HMFSs), rich in palmitic acid (16:0) at the sn-2 position of triacylglycerol (TAG) and rich in unsaturated fatty acids (FAs) (oleic acid, 18:1 and linoleic acid, 18:2) at the sn-1(3) positions, has gained popularity. In this study, HMFSs containing polyunsaturated fatty acids (PUFAs) predominantly at the sn-2 position were prepared, and their oxidation stabilities were compared. First, a non-PUFA-containing HMFS (NP-HMFS) was produced by enzymatic reactions using Novozyme® 435 and Lipozyme® RM-IM as the enzymes and lard as the raw material. Second, HMFSs, containing 10 % PUFA at the sn-2 or sn-1(3) position, were individually prepared by enzymatic reactions using lard and fish oil as raw materials. Here, sn-2-PUFA-monoacylglycerol (MAG) was extracted from the reaction solution using a mixture of hexane and ethanol/water (70:30, v/v) to produce high-purity sn-2-PUFA-MAG with 78.1 % yield. For the PUFA-containing HMFS substrates, comparable oxidation stability was confirmed by an auto-oxidation test. Finally, HMFSs containing 10 % or 2 % sn-1,3-18:1-sn-2-PUFA-TAG species were prepared by enzymatic reactions and subsequent physical blending. The oxidative stability of sn-1,3-18:1-sn-2-PUFA-HMFS was two-fold higher than that of 1/2/3-PUFA-HMFS in which each PUFA was located without stereospecific limitations in TAG. The removal of PUFA-TAG molecular species with higher concentrations of unsaturated units had a significant effect. In addition, the oxidation stability increased with the addition of tocopherol as an antioxidant. Thus, the combined use of two strategies, that is, the removal of PUFA-TAG molecular species with high concentrations of unsaturated units and the addition of antioxidants, would provide a PUFA-containing HMFS substrate with high oxidative stability.

Time Economical Total Synthesis of (-)-Oseltamivir.

A time economical 60 min total synthesis of (-)-oseltamivir was accomplished in a single reaction vessel over five steps. One of the key issues is reduction in the number of steps by eliminating lengthy reaction steps with substitution of a rapid epimerization step. A catalytic system consisting of three reagents, namely, diphenylprolinol silyl ether, thiourea, and acid, was developed for a rapid asymmetric Michael reaction with excellent diastereo- and enantioselectivities. All reactions were optimized in terms of not only yield and selectivity but also reaction time.

A CLN2-related and thermostable serine-carboxyl proteinase, kumamolysin: cloning, expression, and identification of catalytic serine residue.

The gene encoding kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase, was cloned and expressed. (i) Kumamolysin was synthesized as a large precursor consisting of two regions: amino-terminal prepro (188 amino acids) and mature proteins (384 amino acids). (ii) The deduced amino acid sequence of the mature region exhibited high similarity to those of such bacterial pepstatin-insensitive enzymes as Pseudomonas carboxyl proteinase (PSCP; EC 3.4.23.37, identity = 37%), Xanthomonas carboxyl proteinase (XCP; EC 3.4.23.33, identity = 36%), and human CLN2 gene product (identity = 36%), which is related to a fatal neurodegenerative disease. (iii) The presumed catalytic triad, Glu78, Asp82, Ser278 [three-dimensional structure of PSCP: Wlodawer, A. et al. (2001) Nature Struct. Biol., 8, 442-446], was found to be conserved in the amino acid sequence of kumamolysin. (iv) Kumamolysin was inactivated by such aldehyde-type inhibitors as Ac-Ile-Pro-Phe-CHO (K(i) = 0.7 0.14 microM). In PSCP, it has been clarified that these inhibitors form a hemiacetal linkage with the catalytic serine residue and inactivate the enzyme. (v) Mutational analysis of the Ser278 residue revealed that the mutant lost both auto-processing activity and proteolytic activity. These results strongly suggest that kumamolysin has a unique catalytic triad consisting of Glu78, Asp82, and Ser278 residues, as previously observed for PSCP.

Photoelectrochemical sensor with porphyrin-deposited electrodes for determination of nucleotides in water.

A 5,10,15,20-tetra(4-pyridyl)porphyrin (TPyP)-deposited ITO electrode as a sensor of nucleotides using photocurrent change was prepared. The TPyP-deposited ITO electrode could repeatedly detect nucleotides having concentrations of the microM order by a decrease in the photocurrent.

Strigolactones, host recognition signals for root parasitic plants and arbuscular mycorrhizal fungi, from Fabaceae plants.

Both root parasitic plants and arbuscular mycorrhizal (AM) fungi take advantage of strigolactones, released from plant roots as signal molecules in the initial communication with host plants, in order to commence parasitism and mutualism, respectively. In this study, strigolactones in root exudates from 12 Fabaceae plants, including hydroponically grown white lupin (Lupinus albus), a nonhost of AM fungi, were characterized by comparing retention times of germination stimulants on reverse-phase high-performance liquid chromatography (HPLC) with those of standards and by using tandem mass spectrometry (LC/MS/MS). All the plant species examined were found to exude known strigolactones, such as orobanchol, orobanchyl acetate, and 5-deoxystrigol, suggesting that these strigolactones are widely distributed in the Fabaceae. It should be noted that even the nonmycotrophic L. albus exuded orobanchol, orobanchyl acetate, 5-deoxystrigol, and novel germination stimulants. By contrast to the mycotrophic Fabaceae plant Trifolium pratense, in which phosphorus deficiency promoted strigolactone exudation, neither phosphorus nor nitrogen deficiency increased exudation of these strigolactones in L. albus. Therefore, the regulation of strigolactone production and/or exudation seems to be closely related to the nutrient acquisition strategy of the plants.