Quantum dot-insect neuropeptide conjugates for fluorescence imaging, transfection, and nucleus targeting of living cells

We identified an insect neuropeptide, namely, allatostatin 1 from Drosophila melanogaster, that transfects living NIH 3T3 and A431 human epidermoid carcinoma cells and transports quantum dots (QDs) inside the cytoplasm and even the nucleus of the cells. QD-conjugated biomolecules are valuable resources for visualizing the structures and functions of biological systems both in vivo and in vitro. Here, we selected allatostatin 1, Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2, conjugated to streptavidin-coated CdSe-ZnS QDs. This was followed by investigating the transfection of live mammalian cells with QD-allatostatin conjugates, the transport of QDs by allatostatin inside the nucleus, and the proliferation of cells in the presence of allatostatin. Also, on the basis of dose-dependent proliferation of cells in the presence of allatostatin we identified that allatostatin is not cytotoxic when applied at nanomolar levels. Considering the sequence similarity between the receptors of allatostatin in D. melanogaster and somatostatin/galanin in mammalian cells, we expected interactions and localization of allatostatin to somatostatin/galanin receptors on the membranes of 3T3 and A431 cells. However, with QD conjugation we identified that the peptide was delivered inside the cells and localized mainly to the cytoplasm, microtubules, and nucleus. These results indicate that allatostatin is a promising candidate for high-efficiency cell transfection and nucleus-specific cell labeling. Also, the transport property of allatostatin is promising with respect to label/drug/gene delivery and high contrast imaging of live cells and cell organelles. Another promising application of allatostatin is that the transport of QDs inside the nucleus would lift the limit of general photodynamic therapy to nucleus-specific photodynamic therapy, which is expected to be more efficient than photosensitization at the cell membrane or in the cytoplasm as a result of the short lifetime of singlet oxygen.     

Highly diastereo- and enantioselective direct aldol reactions of aldehydes and ketones catalyzed by siloxyproline in the presence of water

Proline-based organocatalysts have been developed for a highly enantioselective, direct aldol reaction of aldehydes and ketones in the presence of water. While several surfactant-proline combined catalysts have proved effective, proline derivatives with a hydrophobic moiety such as trans-siloxy-L-proline and cis-siloxy-D-proline, both of which are easily prepared from the same commercially available 4-hydroxy-L-proline, have been found to be the most effective organocatalysts examined in this study, affording the aldol product with excellent diastereo- and enantioselectivities, these two catalysts generating opposite enantiomers. Water affects the selectivity, and poor results are obtained under neat reaction conditions or in dry organic solvents. More than three equivalents of water are required for the best diastereo- and enantioselectivities, while three equivalents is the recommended amount from a synthetic point of view. The reaction proceeds in the organic phase, and also proceeds in the presence of a large amount of water. The large-scale preparation of aldols with the minimal use of an organic solvent, including in the purification step, is described.     

Synthesis of 6-formylpterin nucleoside analogs and their ROS generation activities in the presence of NADH in the dark

We demonstrated previously that 3-position-modified 6-formylpterin (6FP) derivatives produce reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) from oxygen in the presence of NADH in the dark. It has been shown that 6FP derivatives markedly generate ROS, which gives rise to their particular physiological activities, such as induction of apoptosis in cellular and living systems, suggesting that such compounds provide a hint for the design of a ROS controlling agent in vivo. However, it is not well understood why such unique activities appear on chemical modification. In the present study, in order to see the effect on ROS generation activity in the dark by the modification of the 1-position in 6FP, we have developed a new synthetic procedure for nucleoside analogs of 6FP and prepared 1-(beta-d-ribofuranosyl)-2-(N,N-diethylaminomethyleneamino)-6-formylpteridin-4-one (RDEF) and 1-(beta-d-ribofuranosyl)-2-(piperidine-1-ylmethyleneamino)-6-formylpteridin-4-one (RPIF) in which the 1-position of 6FP is glycosylated. At pH 7.4, NADH was spontaneously oxidized to NAD(+) in the presence of RDEF in the dark. Using electron paramagnetic resonance analysis coupled with the spin trapping technique, we show that O(2) was converted to H(2)O(2)via superoxide anion radical ( O(2)(-)) during this reaction. The modification of the 1-position of 6FP did not cancel ROS generation activities, which were demonstrated in 3-position-modified 6FPs. Since the 6FP derivatives developed in the present study have a ribose moiety, these compounds can be subjected to further derivatization, such as incorporation into oligonucleotides, oligosaccharides, proteins, or any other compounds that recognize and interact with specific biomolecules, and therefore would be useful in pharmaceutical investigations that need generation of appropriate and controllable amounts of ROS in vivo.     

Synthesis of novel polyxanthenes from poly(arylene ether)s containing benzoyl groups

Poly(arylene ether)s ( 3 ), ( 4 ) containing pendant benzoyl groups as precursors for novel polyxanthenes ( 7 ), ( 8 ) were prepared by nucleophilic substitution reaction of 2,5-difluoro-4-benzoylbenzophenone ( 1 ) or 2,5-difluoro-4-(4-dodecylbenzoyl)-4′-dodecylbenzophenone ( 2 ) with hydroquinone derivatives in the presence of potassium carbonate in N,N-dimethylacetamide. The polycondensation proceeded smoothly at 165°C and produced poly(arylene ether)s with inherent viscosities up to 0.80 dL/g. The novel polyxanthenes were synthesized via the reduction of poly(arylene ether)s followed by the Friedel-Crafts cyclization of diol polymers. The structure of the polyxanthenes was characterized by ¹H-NMR and IR spectroscopies. Polyxanthene 8 was quite soluble in chloroform and THF. The 10% weight loss temperature of polyxanthene 7 was 510°C in nitrogen and it was 90°C higher than the corresponding poly(arylene ether) 3 . © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35 : 2267–2272, 1997     

Synthesis and functionalities of poly(N-vinylalkylamide). V. Control of a lower critical solution temperature of poly(N-vinylalkylamide)

N-vinyl-n-butyramide (NVBA), N-vinylisovaleramide (NVIVA), and N-vinyl-n-valeramide (NVVA), which are N-vinylalkylamides with different alkyl groups were synthesized and their solution behavior in a polymeric form was examined. Copolymers of N-vinylisobutyramide (NVIBA) with N-vinylacetamide (NVA), NVIBA with NVVA, and NVVA with NVA were prepared by the solution polymerization to control the LCSTs. The resultant polyNVBA showed a lower critical solution temperature (LCST) sharply at 32°C, but polyN-vinylisovaleramide (polyNVIVA) and polyN-vinyl-n-valeramide(polyNVVA) that have n-butyl and isobutyl groups, respectively, on their side chains were insoluble even in cold water. The water solubility of the resulting polymers was found to vary, depending on the molecular shapes as well as the side chain length of the alkyl groups in question. The copolymers consisting of NVVA, NVIBA, and NVA in water showed LCSTs sharply between 10 and 90°C, depending on changes in their comonomer content. It was found that the changes in LCST that are caused by the incorporation of comonomers are due to changes in the overall hydrophilicity of the polymer. © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35: 3087–3094, 1997     

Studies on thermophile products. V. Immunosuppressive profile in vitro of Bacillus stearothermophilus component, Fr.5-B.

The immunosuppressive profile of Bacillus stearothermophilus UK563 component, Fr.5-B, is presented in in vitro studies. Fr.5-B (0.1-1000 ng/ml), provided it was added at the initiation of mixed leukocyte reaction (MLR), inhibited dose-dependently the incorporation of tritiated thymidine ([3H]TdR) into mouse spleen cells and human peripheral blood lymphocytes. Even the addition of Fr.5-B 48 h after the onset of culture suppressed mouse MLR, unlike cyclosporin A (CYA). Fr.5-B significantly inhibited cytotoxic T lymphocyte generation determined by [3H]TdR-release micro-cytotoxicity assay by using mouse mastocytoma P815 as targets. Moreover, this component decreased dose-dependently the expression of class II major histocompatibility molecules (Ia) on mouse peritoneal macrophages induced by concanavalin A supernatant. The present results revealed the unique immunosuppressive property of Fr.5-B which was different from that of CYA.     

Synthesis of aromatic poly(thioether ketone)

Aromatic poly(thioether ketone)s were prepared by the direct polycondensation of aromatic dicarboxylic acids with aryl compounds containing ether or sulfide structures using phosphorus pentoxide/methanesulfonic acid (PPMA) as a condensing agent and solvent. Polycondensation proceeded smoothly and produced aromatic poly(thioether ketone)s with inherent viscosities up to 0.73 dL/g. The synthesis of substituted aryl ketones by the reaction of substituted benzoic acids with aryl compounds in PMMA was studied in detail to demonstrate the feasibility of the reaction for polymer formation. The thermogravimetry of the aromatic poly(thioether ketone)s showed a 10% weight loss in air and nitrogen at around 450 and 460°C, respectively. © 1992 John Wiley & Sons, Inc.     

A new approach to the synthesis of pyridazino[4,5-c]pyridazinones

The reaction of 5-hydrazinopyridazin-3(2H)-ones 1 with α-keto diester 2 in acetic acid afforded the corresponding 4,6-dihydropyridazino[4,5-c]pyridazin-5(1H)-ones 3 and pyrrolo[2,3-d)pyridazin-4(5H)-ones 4 . Compounds 3 were also obtained from 4-bromo-5-hydrazinopyridazin-3(2H)-ones 8 and 2 under milder conditions. 5-Bromo-4-hydrazinopyridazin-3(2H)-one 9 , the regioisomer of 8b , also reacted readily with 2a to give 4,7-dihydropyridazino[4,5-c]pyridazin-8(1H)-one 10b , the regioisomer of 3b .     

Control of thermoresponsivity of biocompatible poly(trimethylene carbonate) with direct introduction of oligo(ethylene glycol) under various circumstances

Poly(trimethylene carbonate) (PTMC) is a well‐known biodegradable polymer with good biocompatible properties which make it suitable for biomedical applications. Poly(5‐[2‐{2‐(2‐methoxyethoxy)ethyoxy}‐ethoxymethyl]‐5‐methyl‐1,3‐dioxa‐2‐one) (PTMC‐MOE3OM) and copolymers, bearing oligo ethylene glycol (OEG) at the side chain of PTMC backbone, were selected to investigate the cloud point behavior by solvents such as PBS, water, 10% ethanol solution and various ionic strengths. A pH‐responsive copolymer, poly(TMCM‐MOE3OM‐co‐(5‐methyl‐5‐carboxylic‐1,3‐dioxane‐2‐one)) as carboxylic acid carbonate showed a decreased critical temperature at pH 2. Photo‐responsive copolymer, poly(TMCM‐MOE3OM‐co‐coumarin derivatives) bearing 1% and 10% of photo‐induced molecules (7‐[(5‐(5‐methyl‐1,3‐dioxa‐2‐one)methoxy)]‐methoxy coumarin (TMCM‐coumarin)) exhibited a low cloud point because of the hydrophobic moieties. Meanwhile, alternative coumarin polymer including 2% of 4‐methyl‐7‐[(5‐(5‐methyl‐1,3‐dioxa‐2‐one)methoxy)butoxy)]‐methoxy coumarin (TMCM‐4‐methyl‐coumarin) has been successfully synthesized and copolymerized as a novel molecule. The various combinations of monomers were studied and the significant properties were determined via external triggers after copolymerization. This study showed basically synthetic progress toward designs and trivial rationalization of thermoresponsive copolymers close to body temperature. At present, various pendant groups as side part affect to the lower critical solution temperature (LCST) and biodegradable polymer in order to utilize the actual external stimuli application. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 3466–3474     

Optically detected electron—nuclear double resonance and electron—nuclear—nuclear triple resonance study of 17O hyperfine coupling in the lowest nπ* triplet state of benzil

Optically detected ENDOR and electron—nuclear—nuclear triple resonance of ¹⁷O were measured via phosphorescence from ³(nπ^*) benzil in benzophenone-d₁₀ crystals at high magnetic field. The n and π^* spin densities on the oxygen atom are 0.201 and 0.092, respectively, the angle between the two CO bonds being 150°.