Февраль 2015 Пн Вт Ср Чт Пт Сб Вс 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Стыдно четателю, что обычную студенческую работу за свою выдал.
Оно и понятно, уровень то работы так себе, дальше пределов института
не выйдет. Не понимаю как ты четатель собираешься Нобелевкку получать
печатаясь раз в пять лет в кампусных многотиражках.
Vladimir Sidorov• 11.02.2015 07:25
2013 год, валшебнег, да ты сам найдешь. Свежее пока вкидывать не буду, не имею права. Через месяцок в печать выйдет, поделюсь.
Vladimir Sidorov• 11.02.2015 07:24
А, это суперскрипт, валшебнег. Означает, откуда статья происходит. Ниже - описания всех суперскриптов. Звездочка над моей фамилией означает не то, что ты подумал, а то, что я принципиальный автор исследования.
Леня Хлыновский в свое время уже задавал эти вопросы, зря ты не читал. В статье есть два почетных места. Первое - это место аспиранта/пост дока, который бильше всех руками поработал, и последнее, это место - принципиального исследователя, который идею разработал, своих аспирантов работой загрузил, статью написал, и в журнал послал.
Так понятно, мудак?
четатель решил забить эфир английским текстом. Он думает что не видно,
что это обычная студенческая работа, где он приписал себя с женой как
научных руководителей.
Четатель, ты там мало того ,что в соавторстве, так еще и крайним номером, и женской фамилией )))
Судя по дате ссылок этой работе лет пять как минимум или нет? Дата где публикации и место?
Вообще профессора любят падать на хвост студентам талантливым и влезать в соавторство. Гляжу в омерике также.
четатель, где твоя работа без соавторов, или где ты хотя бы первым номером? Ну или где статья в солидном научном журнале.
Vladimir Sidorov• 11.02.2015 07:09
Дальше продолжаем, для дорогого валшебнега:
buffer (100 µM HPTS, 100 mM NaCl or 75 mM Na2SO4, 10 mM sodium phosphate, pH 7.0). During the hydration process the suspension was exposed to 10 freeze-thaw cycles (MeOH/dry ice, 45°C water bath). The resulting suspension was submitted to 21 high-pressure extrusions at room temperature through a 0.1 µm membrane affording liposomes. The liposomes were purified via size-exclusion chromatography (Sephadex G-75 was used as a solid phase, the same buffer as used for the hydration of liposomes except HPTS, was used as an eluent). The liposomes eluted as a cloudy solution that appeared light blue under UV light (2.0 mL). For the incubation and fluorimetric experiments, the collected liposomes were diluted in sodium phosphate buffer (100 mM NaCl, 10 mM sodium phosphate, pH 4-7.0) to a final concentration of 500 µM.
Fluorescence studies of pH-controlled release of liposome contents. In a typical experiment, the suspension of liposomes (10.0 mL at 500 µM concentration of total lipid) in sodium phosphate buffer (100 mM NaCl, 0-10 μM triphenoxyacetamide 3, 10 mM sodium phosphate, pH 4-7.0) was stirred at room temperature. Aliquots of 2.0 mL were analyzed at timepoints of initial, one hour, four hours, 11 hours and 24 hours. All flourometric experiments at each timepoint were kept consistent. The 2.0 mL sample was loaded in a thermostated spectrophotometric cell. The liposome suspension was stirred at 25°C during the course of analysis. The emission of HPTS at 511 nm was monitored with concurrent excitation at 405 and 460 nm. The emission and excitation slits of the spectrophotometer were set at 5 nm, time increments were set at 2.0 s, and integration time was set at 0.5 s. The total time of each experiment was 600 s. An
injection of cyclen tetraurea 4 (20 μL of 1.0 mM solution in DMSO) was made at 60 s, followed by the injection of Triton-X 100 (40 μL of 25% wt/v in Millipore H2O) at 500 s.
Liposome preparation for dynamic light scattering studies. The preparations of liposomes were similar to those for pH-controlled release studies. For these preparations the use of HPTS dye was omitted and liposomes were not passed through a sephadex column. The liposome suspension was transferred from an extruder syringe into a vial, after 21 extrusions. The liposomes were diluted in sodium phosphate buffer (100 mM NaCl, 10 mM sodium phosphate, pH 7.0) to a final conetration of 500 µM in the total lipid.
Dynamic light scattering studies. Liposome suspensions (2.0 mL) were analyzed in a disposable cuvette at 25°C on a Malvern Zetasizer Nano Series Instrument. An initial run was performed to determine the size of the liposomes. Then human serum albumin (HSA, 100 mg) was added to the liposome suspension and the resulting suspension was mixed by inverting. All DLS runs consisted of 10 separate measurements of Z average obtained every four minutes, with the average value being reported for each experiment.
MTS cell proliferation assay. B16F10 tumor cells were employed to evaluate liposomes for effects on cell viability with the MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. MTS assay is a colorimetric method for determining the number of viable cells by measuring the activity of enzymes that reduce MTS in the presence of phenazine methosulfate to formazan,
giving a purple color. After the cells (104 cells/well) were cultured in a 96-well plate for 3 days in the presence of various concentrations of SUVs, MTS solution (Promega) was added and the absorbance at 490 nm was recorded using a 96-well plate reader (Molecular Devices Thermomax Plate Reader, Rockville, MD). Results are presented as the mean ± S.E. Student t-test was used for detection of differences. Transmission images of the cells were obtained on Olympus IX50 microscope (Olympus Inc., Tokyo, Japan), using 20× magnification.
RESULTS AND DISCUSSION
Formulation of liposomes. In order to develop an operational drug delivery system, an appropriate lipid composition in formulations of liposomes had to be constructed. The proper liposomal composition would allow us to accomplish the following: 1) to encapsulate the drug model in liposomes and 2) to release this model upon hydrolysis of acetal groups of lipids 1 or 2. 8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS) was chosen as the drug model (Kano, 1978). When encapsulated in liposomes, this pH sensitive dye can detect internal pH changes through the change in fluorescence of its protonated and deprotonated forms; furthermore, the release of the dye can be monitored through exogenous application of cyclen-tetrathiourea compound 4 (Winschel, 2005). This membrane-impermeable compound binds to HPTS dye in PBS with submicromolar affinity and high selectivity, and quenches 90% of its fluorescence (Winschel, 2005). The typical calcein and carboxyfluorescein release assays (Rex, 1996; Barbet, 1984) cannot be used for these purposes, as a consequence of the high (100 mM) concentration of dye required for the fluorescence self-quenching. At these concentrations, the dyes with pKa’s
of 6.7-7.0 (Aschi, 2008) would act as buffering compounds and preclude acidification of liposomal interior during the uptake of HCl. At the same time, liposomes, loaded with HPTS dye at low (100 μM) concentration can be readily acidified through such co-transport of HCl. A different technique for monitoring of contents release, that relies on the encapsulated mixtures of HPTS and p-xylene-bis-pyridinium bromide (DPX) (Ng, 2009; Van Bambeke, 2000; Morilla, 2005) was also proven unsuitable for our purposes. In this technique, for optimal quenching of the HPTS fluorescence, DPX has to be used at 50 mM concentration, which creates 100 mM internal concentration of bromide anion. Bromide is also a substrate for the HCl co-transporter 3 (unpublished data); therefore, the efflux of HBr from liposomes would nullify the effects produced by the influx of HCl.
Proper lipid composition was determined by monitoring of the extent of HPTS encapsulation. In a typical experiment, a 2 mL aliquot of the stock suspension of liposomes (see experimental) was transferred into a fluorimetric cell, and 10 μL of 1 mM solution of compound 4 in DMSO was injected, followed by the injection of the detergent Triton X-100. The extent of fluorescence quench upon injection of compound 4 was attributed to the portion of the dye remaining outside the liposomes. The extent of fluorescence quenching after the lysis of liposomes with Triton X-100 was due to the dye encapsulated in the aqueous compartments of liposomes and therefore not accessible to compound 4 prior to lysis. We found that neither lipid 1, nor lipid 2 were capable of encapsulating HPTS dye when utilized in their pure forms. Presumably, the two lipids did not form liposomes at all; consequently, a more complex mixture of lipids was used as an alternative.
We found that the most effective encapsulation of HPTS dye occurred for the lipid formulations comprising 25 mol% of EYPC, 25 mol% of DOPE and 50% of lipid 1 or 2.
pH-dependent release of contents. First, we demonstrated that liposomes containing lipids 1 and 2 were actually pH sensitive. To accomplish this, two sets of liposomes formulated with lipid 1 and lipid 2, were prepared in PBS (100 mM NaCl, 10 mM Pi, pH = 7.0, 100 μM HPTS inside). Then, these liposomes were incubated in HPTS-free PBS at pH 4 and 6, respectively, for various periods of time, as detailed in the experimental section. After incubation, liposomes were transferred into a fluorimetric cell, and the fluorescence of HPTS was monitored (ex 405 nm, em 510 nm). During each experiment, a DMSO solution of compound 4 was injected, followed by lysis of liposomes at 500 s.
Liposomes prepared from lipid 2 were stable at neutral pH for only about 1 hour and displayed an instant collapse at reduced pH. However, liposomes prepared from lipid 1 were stable at neutral pH for extensive periods of time and showed pH-dependent contents release at lower pH (figure 1). Thus, at pH 6, liposomes formulated with lipid 1 did not exhibit a significant release of HPTS for the first 24 hours, yet, a rapid release of HPTS from these liposomes was observed in the next 12 hours (figure 1B). The same liposomes, when incubated in PBS at pH 4, were stable for 4 hours, and released their entire contents after 8 hours of incubation. These experiments demonstrated that liposomes prepared from lipid 1 were indeed pH sensitive. A rather quick then gradual release of the dye is consistent with rapid liposome to micelle transition, caused by a buildup of amphiphilic hexadecanol in the membrane of liposomes upon hydrolysis of lipid 1 (Sandstrom, 2005; Barriocanal, 2005).
INSERT FIGURE 1
Release of HPTS from asymmetric liposomes. After revealing that formulations of liposomes containing lipid 1, respond to the external change in pH, we demonstrated that the variations in internal pH could also affect their stability. In order to achieve this task, two populations of liposomes were prepared with an identical lipid composition (25 mol% DOPE, 25 mol% EYPC and 50 mol% lipid 1) but various aqueous internal contents. The first set was formulated with PBS (100 mM NaCl, 10 mM Pi, pH = 7, 100 μM HPTS), and the second set of liposomes was formulated with sodium sulfate instead of sodium chloride (75 mM Na2SO4, 10 mM Pi, pH = 7, 100 μM HPTS). Seventy-five mM Na2SO4 was used in place of 100 mM NaCl to ensure isoosmolality of the internal solution of liposomes to that of the external PBS (Sidorov, 2002).
Both populations of liposomes were suspended in PBS followed by the subsequent addition of 10 μM compound 3. Liposomes containing Na2SO4 demonstrated rapid acidification of their internal compartments to a pH of approximately 5.6, as evidenced by change in the fluorescence of HPTS (figure 2A, lower trace) (Kano, 1978). After acidification, the internal pH did not change significantly until liposomes were lysed with Triton X-100, after a period of 1 hour. A small increase in the internal pH (~ 0.1 pH units) observed during this time was most likely due to insignificant decomposition of liposomes and subsequent release of the dye. Lysis of liposomes resulted in a complete release of HPTS into the external buffer, which caused the increase of pH back to 7. Application of compound 3 to the suspension of NaCl-containing
liposomes did not produce a change in the internal pH due to the lack of Cl- gradient across the bilayer membrane (figure 2A, upper trace).
INSERT FIGURE 2
After verifying that liposomes that are under a Cl- gradient can be acidified by application of triphenoxyacetamide 3, we incubated both populations of liposomes in PBS for various periods of time. At the end of each period, the extent of contents release was analyzed fluorimetrically (figure 3). The symmetrical liposomes (NaClin/out) did not show substantial release of the dye for at least four days, but liberated all their contents after 6.5 days. At the same time, asymmetrical liposomes (Na2SO4in/NaClout) were stable for only 13 hours, and released their contents almost completely after 2.5 days.
INSERT FIGURE 3
Finally, we decided to prepare a mixture of both populations of liposomes and consequently analyze the contents release using this model (figure 4). As can be seen, the release of contents illustrated an intricate profile, wherein the periods of inactivity were followed by rather rapid release of HPTS. A plateau at ~96 hours of incubation corresponds to the time segment, where the entire contents of the asymmetrical subpopulation of liposomes was released, and the symmetrical liposomes did not begin to decompose yet. This observation suggests that for the preferred concentration of liposomal suspension, there was no transfer of hexadecanol from the micelles into the intact liposomes, and no hexanol-induced fusion of the hydrolyzed and intact membranes took place; two populations of liposomes released their contents independently from each other. This observation is similar to that reported for the oleoyl alcohol effects on the
stability of pH-sensitive liposomes (Sudimack, 2002). Although the initial period of inactivity for the aforementioned mixture of liposomes was approximately12 hours, a faster release of the contents could be achieved through addition of a more flexible and hence more pH-sensitive lipid 2 into the formulation of liposomes.
INSERT FIGURE 4
Biocompatibility. Although this work did not involve any in vivo studies, the principal biocompatibility of these pH-sensitive liposomes was demonstrated. It is known that liposomes with a net positive charge on their surface are rapidly cross-linked with serum albumin within blood plasma and then cleared from circulation (Li, 2007; Li, 1998). Dynamic light scattering (DLS) studies can be used for monitoring of the relative size of particles in the presence of human serum albumin (HSA) (Uhl, 2007; Ayame, 2008; Sabin, 2009). We performed a series of DLS experiments on four types of liposomes. Two sets were formulated using lipids 1 and 2, and two other sets were formulated with cationic precursors 1c and 2c. Consistent with the zwitterionic nature of lipids 1 and 2, and cationic nature of lipids 1c and 2c, the four sets of liposomes exhibited surface zeta potentials of -0.2±0.1, +0.2±0.1, +22.6±0.4 and +26±0.6 (mV), respectively. The two latter formulations were used as positive controls, since they were expected to change in size upon interactions with HSA.
In the absence of HSA all four formulations of liposomes were approximately 100 nm in diameter (figure 5 A), consistent with the extrusion of liposomes through 100 nm pores. However, incubation of these liposomes with HSA for 40 minutes resulted in a formation of particles of varying sizes in all four experiments. Small size particles of about 6 nm were observed immediately upon addition of HSA, which is consistent with
the size of human serum albumin globules (Kiselev, 2001). Larger size particles with the diameter of 300-1000 nm were detected immediately upon addition of HSA to the suspensions of liposomes formulated with cationic lipids 1c and 2c (Figure 5B, two bottom traces). No such immediate formation of larger particles was observed during the first 20 minutes for lipids 1 and 2. However, a small amount of larger aggregates with the diameter of ~ 800 nm became detectable for lipid 2 after 40 minutes (figure 5B, third trace from the bottom). Formulation with lipid 1 did not undergo any noticeable changes during this time (figure 5B, upper trace). These results suggest that at least liposomes formulated with lipid 1 do not form large aggregates in the presence of HSA, and therefore potentially suitable for the in vivo applications.
INSERT FIGURE 5
Cytotoxicity of liposomal formulations. To show general non-toxicity of liposomal formulations, cell viability assays were conducted with liposomes, formulated with lipid 1 and compound 3. Since compound 3 is a HCl co-transporter, it was possible that this compound exhibits certain cytotoxicity as other natural HCl co-transporters (e.g. prodigiosin) (Francisco, 2007; Montaner, 2003). However, when B16F10 tumor cells were incubated with the synthetic liposome mixture, at concentrations exceeding 1 mM (total lipid concentration) and compound 3 at 20 μM concentration, cell proliferation was indistinguishable from the negative control (figure 6 A). In contrast, the positive control
experiment, in which the cells were incubated with a potent cytotoxin curcumin, (Woo, 2003; Donatus, 1990) resulted in virtually complete cell death, signifying that the cell culture responded to the cytotoxic substances. No morphological signs of cell change or death were observed upon treatment of B16F10 tumor cells with the aforementioned liposomes (Wei, 1998) (figure 6 B).
INSERT FIGURE 6
CONCLUSION
In conclusion, we developed a new controlled drug release system that is based on a mixture of several populations of pH-sensitive liposomes. Unlike other pH-responsive drug delivery systems that required exogenous acidification for release, this system was initiated through the acidification of the internal aqueous compartments of liposomes upon the contact with physiologically relevant solution of NaCl. A degree of such acidification was controlled by the internal concentrations of NaCl and allowed to release the contents of liposomes in 12 hours through 7 days. The drug delivery system, based on lipid 1 was shown to be not sensitive to the physiological concentrations of HSA, and non-toxic at total lipid concentrations of up to 1 mM and concentrations of compound 3 up to 20 μM. Our future studies will focus on extension of the time range for the controlled drug release, as well as on the in vivo applicability of this new drug delivery
Vladimir Sidorov• 11.02.2015 07:07
Дорогой валшебнег, конечно, почитай:
Controlled Drug Release System Based on pH-Sensitive Chloride-Triggerable Liposomes
Mark P. Wehunta, Christine A. Winschela, Ali K. Khana, Tai L. Guob, Galya R. Abdrakhmanovac and Vladimir Sidorova*
aVirginia Commonwealth University, Department of Chemistry, 1001 W. Main St. Richmond VA, 23284
bVirginia Commonwealth University, Department of Pharmacology and Toxicology, 527 N. 12th St., Strauss Research Laboratory, Richmond VA, 23298
cDepartment of Pharmacology and Toxicology, Virginia Commonwealth University, 1112 E.Clay Mcguire Hall Annex, Richmond, VA 23298
*Corresponding author. Email: vasidorov@vcu.edu Phone: (804)-828-7507
ABSTRACT
New pH-sensitive lipids were synthesized and utilized in formulations of the liposomes suitable for controlled drug release. These liposomes contain various amounts of NaCl in the internal aqueous compartments. The release of the drug model is triggered by an application of HCl co-transporter and exogenous physiologically relevant NaCl solution. HCl co-transporter allows an uptake of HCl by liposomes to the extent, proportional to the transmembrane Cl- gradient. Therefore, each set of liposomes undergoes internal acidification, which ultimately leads to the hydrolysis of the pH-sensitive lipids and contents release at the desired time. The developed system releases the drug model in a stepwise fashion with the release stages separated by periods of low activity. These liposomes were found to be insensitive to physiological concentrations of human serum albumin and to be non-toxic to cells at concentrations exceeding pharmacological relevance. These results render this new drug release model potentially suitable for in vivo applications.
Biocompatible pH-sensitive liposomes have recently become attractive vehicles for intracellular drug (Ishida, 2001; Turk, 2002) and gene delivery (Li, 2005; Simoes, 2004; Slepushkin, 1997; MacKay, 2008; Chen, 2007). Targeted delivery of pH-sensitive liposomes to tumors, or infected sites with increased acidity was also proven successful (Torchilin, 1993; Mastrobattista, 2002). Whereas the reports on applications of pH-sensitive liposomes to the intracellular delivery of therapeutic components are quite numerous, there are only few reports on the applicability of pH-sensitive liposomes for controlled drug release (Ng, 2009; Jain, 2007; Couffin-Hoarau, 2004; Tang, 2008). The
lack of applications of pH-sensitive liposomes for controlled drug release in blood plasma is due to the narrow range of intravascular pH, which is largely maintained by carbonic anhydrase (Pocker, 1967). All literature precedence concerning pH sensitive liposomes reports a release of contents via an exogenous change in pH; and, to the best of our knowledge, a release of contents due to an internal liposome acidification has yet to be reported. The internal acidification of liposomes, triggered by contact with blood plasma or other physiological fluid, provides a new model of a controlled drug release system, suitable for therapy of diseases such as diabetes (Heinemann, 2001).
In this paper, we report on the development of a new drug delivery system that provides a release of the drug model through a series of burst-like events. The system is based upon the mixture of several sets of liposomes containing pH-sensitive lipids 1 or 2 in the bilayer membrane and various amounts of NaCl in the internal buffers. The release of contents of liposomes is triggered by contact with phosphate buffer saline (PBS) and/or application of HCl co-transporter 3 (scheme 1) (Sidorov, 2003).
INSERT SCHEME 1
The HCl co-transporter 3 delivers HCl into the internal aqueous compartments of liposomes, making them more acidic. Since the induced pH change is a function of the Cl- gradient, formulating liposomes with a preset internal NaCl concentration allow their future acidification to specific levels, ultimately causing a kinetically controlled decay of acetals 1 or 2; with the decay occurring fastest in the most acidic liposomes (scheme 2). The acetal-bearing molecules have previously been used in pH-sensitive drug delivery
vehicles, and the kinetics of their hydrolysis makes them suitable for the controlled contents release (Gillies, 2005; Garripelli, ; Kaihara, 2009).
INSERT SCHEME 2
EXPERIMENTAL PROCEDURES
General. The 1H NMR spectra were recorded on a Varian AS400 instrument operating at 400.130 MHz or Varian Mercury instrument operating at 299.865 MHz. Chemical shifts are reported in ppm relative to the residual protonated solvent peak. The 13C NMR spectra were recorded on the same instruments at 100.613 MHz and 75.408 MHz respectively and chemical shift values are reported in ppm relative to the solvent peak. Both 1H and 13C spectra were taken in the same solvent and on the same instrument for each sample. The low resolution mass spectra were recorded on a Micromass Q-TOF™ 2 instrument (Manchester, UK) using the electrospray technique (positive mode). The samples were introduced into mass spectrometer using a flow rate of 10 μL/min, the needle voltage was set at 3500 V with ion source at 110 oC and the cone voltage at 35 V. The high resolution mass spectra were recorded on a LCMS-IT-TOF (Shimadzu Corporation, Columbia MD) instrument using the electrospray technique (positive mode). All spectrophotometric experiments were carried out on a Fluoromax 3 (Jobin-Yvon/Horriba) spectrophotometer. The dynamic light scattering analysis of the particle size and surface zeta-potential in liposome suspensions was carried out on the Malvern instrument (Malvern Zetasizer Nano Series, Malvern Instruments). The pH of solutions was monitored with a Corning 350 pH/ion analyzer, using a Ag/AgCl pH-sensitive electrode (Accumet). Chromatography was performed using 60-200 mesh silica purchased from Baker and 40-120 μm Sephadex G-75 purchased from Aldrich. Thin
layer chromatography was performed on Kieselgel 60 F254 and Uniplatetm Silica Gel GF silica-coated glass plates and visualized by I2. High-pressure extrusion was performed on the AvantiTM mini-extruder with a 0.1 μm polycarbonate membrane. All chemicals and solvents were purchased from Aldrich, Sigma or Fluka. Phospholipids were purchased from Avanti Polar Lipids.
Synthesis. Compounds 1 and 2 were synthesized according to scheme 3.
INSERT SCHEME 3
1-(2-bromo-1-(hexadecyloxy)ethoxy)hexadecane 1a. To a solution of p-toluenesulfonic acid-monohydrate (440 mg, 2.58 mmol) in acetone (200 mL) was added a small amount of Na2SO4. The resulting solution mixture stirred at room temperature for 30 minutes and was subsequently filtered to remove remaining Na2SO4. The solvent of the filtrate was removed under diminished pressure affording a white residue. The white residue was mixed with anhydrous THF (25 mL) and transferred to an addition funnel containing bromoacetaldehyde-diethyl acetal (5.0 g, 0.0258 mol) in anhydrous THF (100 mL). The resulting solution mixture was added dropwise over 45 minutes to hexadecanol (61.9 g, 0.255 mol), under N2 at 65°C. The reaction mixture was stirred at reflux (68.8°C) for 3 days. Then, the reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was extracted with hexanes (250 mL) and cooled in an ice bath (0°C) to crystallize out any unreacted hexadecanol, which was filtered off. The remaining organic filtrate was washed with saturated sodium bicarbonate and brine. The organic portions were combined, dried over MgSO4 and
concentrated under diminished pressure producing an orange solid. The crude solid was purified via flash column chromatography (silica gel, 20% CHCl3/Hexanes) affording a white solid (2.009 g, 0.0258 mol, 13%). 1H NMR (300 MHz, CDCl3) δ 4.16 (td, J = 6.7, 0.8 Hz, 1H), 3.60 (d, 2H), 3.37 (t, 4H), 1.65 (dd, J = 14.2, 6.9 Hz, 2H), 1.54 (s, 4H), 1.26 (s, 50H), 0.88 (t, J = 6.7 Hz, 6H). 13C NMR (400 MHz, CDCl3) δ 101.67, 67.18, 32.16, 29.93, 29.60, 26.34, 22.93, 14.35. MS (ESI, high-resolution) ([M+Na]): 613.3284, calcd for C34H69BrNaO2 m/z: 613.44.
2-(2,2-bis(hexadecyloxy)ethoxy)-N,N-dimethylethanamine 1b. NaH (60% in mineral oil, 0.284 g, 0.0118 mol) was carefully mixed with hexanes (100 mL, deoxygenated) at room temperature to separate NaH from mineral oil. After NaH settled, hexanes were removed by gravity filtration. Anhydrous THF (20 mL) was added to the NaH and the resulting solution mixture was cooled to 0°C. A solution of 2-(dimethylamino) ethanol (0.26 mL, 0.0258 mol) was added dropwise to the NaH solution. The resulting solution stirred at 0°C under N2 for 30 minutes. The solution was warmed to room temperature and heated to reflux (70 °C). Compound 1a (0.503 g, 0.852 mmol) in anhydrous THF (50 mL) was then added. The resulting reaction mixture stirred at reflux (70°C) for 3 days. The reaction mixture was slowly cooled to room temperature and subsequently cooled to 0°C. A dropwise addition of H2O, to quench any remaining NaH, was continued until gas evolution ceased. The reaction mixture was extracted with hexanes, washed with brine and concentrated under diminished pressure producing a yellow oil. The crude oil was purified via flash column chromatography (silica gel, 7% MeOH/CHCl3 – 20% MeOH/CHCl3) affording a yellow oil (0.031 g, 6%). 1H NMR (300
MHz, CDCl3) δ 4.61 (t, J = 5.2 Hz, 1H), 3.90 – 3.21 (m, 8H), 2.57 – 2.41 (m, 2H), 2.27 (s, 6H), 1.65 (dd, J = 14.2, 6.9 Hz, 2H), 1.54 (s, 4H), 1.26 (s, 50H), 0.88 (t, J = 6.7 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ 101.66, 71.85, 69.85, 67.14, 58.94, 45.97, 32.42, 32.15, 30.59, 29.84, 29.15, 26.50, 22.91, 14.33. MS (ESI, high-resolution) ([M+1]+): 598.6648, calcd for [C38H79NO3]+ m/z: 598.61.
N-(2-(2,2-bis(hexadecyloxy)ethoxy)ethyl)-2-ethoxy-N,N-dimethyl-2-oxoethanaminium 1c. To a solution of 1b (0.015g, 0.0251 mmol) in diethyl ether (4.0 mL) was added ethylbromoacetate (0.040 g, 0.240 mmol). The reaction mixture was stirred at room temperature for 19.5 hours. The crude reaction solution was concentrated under diminished pressure. The crude residue was purified via column chromatography (silica gel, 10 % MeOH/CHCl3) affording a white solid (0.017g, 0.0248 mmol, 100%). 1H NMR (300 MHz, CDCl3) δ 4.85 (s, 2H), 4.54 (t, J = 4.7 Hz, 1H), 4.22 (dt, J = 10.8, 5.8 Hz, 4H), 3.99 (d, J = 4.3 Hz, 2H), 3.70 (s, 6H), 3.64 – 3.24 (m, 4H), 1.70 (d, J = 18.4 Hz, 4H), 1.58 – 1.39 (m, 4H), 1.39 – 1.21 (m, 50H), 1.00 (dd, J = 6.7, 3.6 Hz, 3H), 0.87 (t, J = 6.7 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ 164.99, 101.66, 71.85, 69.85, 67.14, 58.94, 45.97, 36.32, 32.42, 32.15, 30.59, 29.84, 29.15, 26.50, 22.91, 14.33. MS (ESI, high-resolution) ([M]+): 684.4030, calcd for [C42H86NO5]+ m/z: 684.65.
2-((2-(2,2-bis(hexadecyloxy)ethoxy)ethyl)dimethylammonio)acetate 1. A solution of KOH (0.8 mg, 0.0146 mmol) in H2O (0.3 mL) was added to a solution of 1c (0.002 g, 0.00292 mmol) in THF:MeOH (1:1, 1.0 mL).. The resulting reaction mixture was stirred at room temperature for 15 minutes, then the solution was concentrated under reduced
pressure. The crude material was mixed with CH2Cl2 (2.0 mL) and then subsequently washed with H2O (2.0 mL). The organic layer was concentrated under reduced pressure to give a white solid (1.9 mg, 0.00292 mmol), 100%). 1H NMR (300 MHz, CDCl3) δ 4.52 (m, 1H), 3.89 (d, J = 18.5 Hz, 2H), 3.75 – 3.26 (m, 14H), 1.76 (m, 4H), 1.76 – 0.73 (m, 54H), 1.04 – 0.73 (m, 6H). 13C NMR (75 MHz, CDCl3) δ 165.06, 101.02, 71.48, 67.26, 66.05, 63.24, 53.32, 32.15, 30.68, 29.46, 26.39, 22.92, 14.35. MS (ESI, high-resolution) ([M+H]+): 656.4811, calcd for [C40H82NO5]+ m/z: 656.62.
(Z)-1-(2-bromo-1-((Z)-octadec-9-en-1-yloxy)ethoxy))octadec-9-ene 2a. To a solution of p-toluenesulfonic acid-monohydrate (476 mg, 2.5 mmol) in acetone (200 mL) a small amount of Na2SO4 was added. The resulting mixture was stirred at room temperature for 30 minutes and was subsequently filtered to remove Na2SO4. The solvent of the filtrate was removed under diminished pressure affording a white residue. The white residue was mixed with anhydrous THF (25 mL) and transferred to an addition funnel containing bromoacetaldehyde-diethyl acetal (3.0 g, 0.015 mol) in anhydrous THF (100 mL). The resulting solution mixture was added dropwise over 45 minutes to oleyl alcohol (4.0 g, 0.15 mol), under N2 at 65°C. The reaction mixture was stirred at reflux (68.8°C) for 3 days. Then the reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was extracted with hexanes (250 mL), washed with saturated sodium bicarbonate and brine. The organic portions were combined, dried over MgSO4 and concentrated under reduced pressure producing an orange colored oil. The crude oil was purified via column chromatography (silica gel, 20% CHCl3/Hexanes – 50% CHCl3/Hexanes) affording a lemon colored oil (7.26 g, 0.011 mol, 75%). 1H NMR (400 MHz, CDCl3) δ 5.51 – 5.28 (m, 4H), 4.65 (t, J = 5.4 Hz,
1H), 3.72 – 3.43 (m, 4H), 3.38 (t, J = 9.8 Hz, 2H), 1.99 (dd, J = 13.3, 6.7 Hz, 8H), 1.58 (dd, J = 14.5, 6.8 Hz, 6H), 1.25 (dd, J = 21.7, 9.4 Hz, 42H), 0.88 (t, J = 6.8 Hz, 6H); 13C NMR (300 MHz, CDCl3) δ 130.07, 101.31, 66.67, 32.35, 31.57, 30.22 , 28.42, 26.94, 25.94, 22.43, 13.87. MS (ESI, high-resolution) ([M+Na]+): 663.4293, calcd for C38H73BrNaO2 m/z: 663.47.
2-(2,2-bis((E)-octadec-9-en-1-yloxy)ethoxy)-N,N-dimethylethanamine 2b. Sodium hydride (60% in mineral oil, 2.68 g, 0.11 mol) was carefully mixed with hexanes (100 mL, deoxygenated) under N2 at room temperature to separate NaH from mineral oil. After NaH settled, hexanes were removed via syringe and isopropanol was used to quench any NaH remaining in the removed hexanes solution and syringe. Anhydrous THF (20 mL) was added to the NaH and the resulting solution mixture was cooled to 0°C. A solution of 2-(dimethylamino) ethanol (1.87 mL, 0.019 mol) was added dropwise to the NaH solution. The resulting solution was stirred at 0°C under N2 for 30 minutes. Then the solution was warmed to room temperature and heated to reflux (68.8 °C). A solution of 2a (4.0 g, 0.0062 mol) in anhydrous THF (150 mL) was then added dropwise over a period of 30 minutes. The resulting reaction mixture stirred at reflux (68.8°C) for 4 days. The reaction mixture was slowly cooled to room temperature and subsequently cooled to 0°C. Then, water was added dropwise to quench any remaining NaH, until gas evolution ceased. The reaction mixture was extracted with hexanes, washed with brine and concentrated under reduced pressure producing a brown oil. In some cases, the crude material was recrystallized from ethyl acetate and ethanol.Final purification was carried out via column chromatography (silica gel, 20% CH3CN/CHCl3, 5% MeOH/CHCl3 –
15% MeOH/CHCl3) affording a brown oil (1.43 g, 36%). 1H NMR (400 MHz, CDCl3) δ 5.46 – 5.26 (m, 4H), 4.61 (t, J = 5.2 Hz, 1H), 3.61 (dt, J = 8.9, 6.2 Hz, 4H), 3.57 – 3.40 (m, 4H), 2.55 (t, J = 5.6 Hz, 2H), 2.31 (s, 6H), 1.99 (dd, J = 13.4, 6.8 Hz, 8H), 1.56 (dd, J = 13.9, 6.8 Hz, 4H), 1.27 (d, J = 8.3 Hz, 44H), 0.88 (t, J = 6.7 Hz, 6H). 13C NMR (300 MHz, CDCl3) δ 130.05, 101.08, 71.30, 69.00, 66.65, 58.31, 45.37, 32.34, 31.63, 29.74 , 28.60, 26.93, 25.89, 22.42, 13.86. MS (ESI, high-resolution) ([M+1]+): 650.6887, calcd for [C42H83NO3]+ m/z: 650.64.
N-(2-(2,2-bis((Z)-octadec-9-en-1-yloxy)ethoxy)ethyl)-2-ethoxy-N,N-dimethyl-2-oxoethanaminium 2c. To a solution of 2b (250 mg, 0.384 mmol) in THF (25 mL) ethylbromoacetate (0.128 mL, 1.15 mmol) was added. The resulting reaction mixture was stirred under reflux (68.8°C) for 3 days. The reaction mixture was cooled to room temperature and solvent was removed under reduced pressure. Minor product removed as a solid via recrystallization from ethanol/ethyl acetate. The filtrate was concentrated under reduced pressure producing sticky brown oil. The oil was washed with a small amount of diethyl ether to remove any excess ethyl bromoacetate to give 217 mg, (0.294 mmol) of 2c. 1H NMR (400 MHz, CDCl3) δ 5.36 (d, J = 14.1 Hz, 4H), 4.87 – 4.37 (m, 5H), 3.77 – 3.35 (m, 16H), 2.20 (m, 8H), 1.89 – 1.03 (m, 51H), 0.95 – 0.51 (m, 6H). 13C NMR (300 MHz, CDCl3) δ 156.80, 130.05, 101.08, 69.00, 67.52, 65.87, 62.76, 60.42, 50.87, 49.20, 33.95, 31.92, 29.74, 28.60, 26.93, 25.89, 22.42, 15.39, 13.86. MALDI-TOF/MS ([M]): 736.9, calcd for [C46H90NO5]+ m/z: 736.68.
2-((2-(2,2-bis((Z)-octadec-9-en-1-yloxy)ethoxy)ethyl)dimethylammonio)acetate 2. To a solution of 7 (210 mg, 0.285 mmol) in MeOH:THF:Brine (1:1:1) was added KOH (0.096 g) at room temperature. The reaction mixture was sonicated for 30 minutes, filtered and concentrated under diminished pressure affording a beige colored oil (165 mg, 0.233 mmol). 1H NMR (300 MHz, CDCl3) δ 5.15 (ddt, J = 42.0, 12.2, 6.1 Hz, 4H), 4.92 – 4.75 (m, 2H), 4.63 (d, J = 36.1 Hz, 1H), 4.37 – 3.43 (m, 18H), 3.07 – 1.89 (m, 4H), 2.84 – 1.89 (m, 4H), 2.21 – 1.64 (m, 4H), 1.76 – 1.32 (m, 12H), 1.44 – 1.32 (m, 6H), 1.44 – 0.25 (m, 29H), 0.89 (t, J = 6.7 Hz, 6H). 13C NMR (300 MHz, CDCl3) δ 156.80, 130.05, 101.08, 63.11, 50.90, 49.20, 33.97, 31.91, 29.74, 28.60, 26.93, 25.89, 22.42, 15.39, 13.86. MALDI-TOF/MS ([M+H]): 708.9, calcd for [C44H86NO5]+ m/z: 708.65.
Triphenoxyacetamide 3 was synthesized according to the literature procedure (Sidorov, 2003).
Cyclen tetraurea 4 was synthesized as described (Winschel, 2005).
Liposome preparation for pH-controlled release studies. Egg yolk L-R-phosphatidylcholine (EYPC, ethanol solution, 27 µL, 6.58 µmol), 1,2-Dioleoyl-Sn-Glycero-3-Phosphoethanolamine (DOPE, ethanol solution, 500 µL, 6.72 µmol), and lipid 1 or lipid 2 (solution in CHCl3/MeOH/EtOH, 435 µL, 14.1 µmol) were combined and dissolved in CHCl3 (5 mL), MeOH (0.5 mL) and EtOH (0.5 mL), then concentrated under reduced pressure in the round-bottom flask to produce a transparent film (25 % EYPC, 25% DOPE, 50% lipid 1 or lipid 2). The film was dried under high vacuum for 4 hours, and was subsequently hydrated for 1 h in 800 µL of HPTS sodium phosphate
Интересно, а четатель такой же хреновый химик, как и экономист...
четатель, ты где последний раз публиковался и когда? Где это можно почитать?
Vladimir Sidorov• 11.02.2015 06:48
А чего его пасти? Его подержать нужно, а потом зажарить, гы-гы!
Большего подарка мне Россия в принципе сделать не могла, чем перевести все расчеты на золото. БЫГЫГЫ!
Vladimir Sidorov• 11.02.2015 04:48
Че говорите, золотом теперь только рассчитываться будем? Ай, хорошо!!!
Давайте немножко посчитаем. У Америки золота более 8000 тонн, у России - 1200 тонн. При нынешней цене золота в 1230 долларов за унцию, это, соответственно - на 320 миллиардов золота у Америки, и на 48 миллиардов у России. А какими бюджетами страны оперируют? 2 триллиона у России, 18 триллионов у Штатов. То есть, нужно будет 320 миллиардов превратить в 18 триллионов, а 48 - в 2. Не, я ничо против не имею. У меня золота сейчас на несколько десятков тысяч. Когда оно в 50 раз подорожает, я буду очень рад.
нунепомнюялогин• 11.02.2015 04:26
А чего меня поддерживать. Я тебе, Гексогеныч, не доллар какой-нибудь. Не падаю. И это... мой анти18смвирус меня никуда на твои ссылки не пускает.
ераплан атамный фантамас• 11.02.2015 03:31
нагрузил баха на ай-подик, щас ехал по вечерней америке - как в кинофильме тарковсково как-бутто
Про Русский патриотизм. Почти как Вымя, но своими словами. https://www.youtube.com/watch?v=FfMxfFUpF7c
нунепомнюялогин• 11.02.2015 01:56
любопытный
Мне моя версия больше нравицца. оставьте меня в моем невежестве, плиз!
Яна,
та ну шо це вы... В 44-м году как раз бакс к золоту привязали. 1$=0.9 г золота. Потом де Голль отвёз мешок баксов пароходом на их родину, вернулся с мешком золота. Тут и всё. 1971.
нунепомнюялогин• 11.02.2015 01:48
Кста, мне что больше всего понравилось в той статье, так это как раз утверждение о том, что та пресловутая схема 1944 года и похоронила впоследствие СССР. А тиран Путин отомстил! Ох, какая прелесть.
Янночка,
не перебоянивай мой ночной перевод по твоей ссылке.
любопытный ни дурак)))
нунепомнюялогин• 11.02.2015 01:27
любопытный
Оказываецца, в 1944 году Штаты провернули некую махинацею и ввели в действие финансовую схему оплаты долларом, минуя золото. как они говорят "закрыли окно". И весь мир, як котята, исправно перешел на зеленые нефтедоллары. А в 2014 году некто В.В.Путин тихой сапой взял да "открыл" тое самое окошко. Типо, плевали мы (то есть, вы) на ваши доллары, а ну гоните-ка золотом. В России уже фактически 55 тонн золотого запаса. А бамашки зиленовые скоро... скоро... ну вот и кирдык. Поал?
Яна,
с моей? Нас тут не стояло.
/ Понедельник, 9 Февраля 2015
06:00:14 в Комментарии и
обсуждения
А Путина сегодня по всем каналам
величают "тираном 21га века".
Нагнетают, да./
нунепомнюялогин• 11.02.2015 01:07
любопытный
Да я тока с твоей подачи и узнала про "букет" путинских болячек. Шо за газеты вы читаете несерьезные. И опровергают!
Авотхуй...
http://russian.rt.com/article/73526
Авотещё...
http://russian.rt.com/article/73532
АвотЯне:
http://politikus.ru/articles/42840-amerikanskiy-zhurnalist-obyasnil-pochemu-chertov-zapad-obrechen.html
Обама пригрозил Путину по телефону http://tvzvezda.ru/news/vstrane_i_mire/content/201502110003-jb77.htm
Председатель Европарламента: Америка не должна вмешиваться в конфликт на Украине http://www.youtube.com/watch?v=ocnYQ_gsWbc
ОБСЕ: обстрел Краматорска усугубляет ситуацию во время переговоров http://bit.ly/1zTBNwM
Штаб ДНР: ополченцы не смогли бы попасть по Краматорску
http://ria.ru/world/20150210/1046989466.html
СМИ Италии: в случае провала переговоров Киев «пойдет на дно» первым http://bit.ly/1AhymSx
Дейнего: Переговоры контактной группы в Минске продолжатся утром http://russian.rt.com/article/73585
"Кстати, бабушка моя почему-то слово "Дебальцево" не выговаривала, у неё получалось "Гибальцево""
Фря
Эт, что. Кажется при Ющенко. Точно, при Ющенко. Появилась такая мулька.
Ну вы помните, когда Вместо фамилии "Скворцов" стали записывать во всех документах "Шпак", к примеру. И вот моя тёща стала получать пенсию на фамилию "Гежова". Сразу она конечно не обратила внимание, платят пенсию, да и ладно. Но червь сомнения сделал своё дело.
Что за фигня?! Муж Ежов, сын Ежов, а она Гежова?! Недоумевала. Пошла в пенсионный собес с тем же вопросом ругаться.
Ну, её внимательно выслушали, пообещали разобраться. (это она нам рассказывала)
Ага, разберутся они, - говорю я ей. Бюджетные денюжки, отчетность по ведомостям и статистике, разберутся)))
Так бедная старушка Гежовой и померла. Негодяи.
/Участники контактной группы в Минске взяли перервыв/
/В Минске закончилась двухчасовая встреча трехсторонней контактной группы по урегулированию ситуации на востоке Украины./
Командир украинского батальона «Львов» погиб под Дебальцево http://vz.ru/news/2015/2/10/728943.html
Штаб ДНР: группировку ВС Украины в Дебальцево покинули все командиры http://bit.ly/1AhqJLO
ДНР: О прекращении огня говорить пока рано, участники контактной группы изучают предложенный проект протокола http://russian.rt.com/article/73579
Представитель ЛНР Владислав Дайнего: Стабильный мир без политических договоренностей невозможен. ВИДЕО http://lifenews.ru/news/149685
Участники контактной группы в Минске взяли перервыв http://rusnovosti.ru/posts/363525
валшебнег
Что-то они много врут насчет этого обстрела. То говорили, что боеприпасы взорвались, то торнадой из под Горловки ударили, то теперь говорят, что это со стороны Луганска была атака. Брешут.
Ну зато сейчас вся прогрессивная общественность с подачи укро СМИ переживает обстрел ополченцами аэропорта в Краматорске.
Как в декабре не будет? Ну-ну, в городе целых стекол практически не осталось, как и целых газопроводов, школ, больниц, шахт...
Ваше имя
Это характерно для украиноязычных, буквы "ф" в украинском нет, приходится говорить хвиртка, хвартук, хвист опять же.
"у неё получалось "Гибальцево""
А местные жители Фащевки паталогически не выговаривают букву "ф". Только "хв" - "конхвета", "бухвет", "Хващевка".
"Опять перемирие для передислокации?"
Ну, вряд ли. Сейчас показывали кусочек с этих переговоров - Пушилин и Дейнеко выбегали по быстрому к журналистам, настроены решительно, говорят "так, как в декабре, не будет".
- Представители самопровозглашенных ДНР и ЛНР передали во вторник вечером участникам контактной группы проект протокола по мирному урегулированию ситуации, сложившейся в Донбассе.
"Мы передали участникам трехсторонней контактной группы проект протокола, включающий комплекс мер по военному и политическому урегулированию. Стороны контактной группы взяли данный проект и обещали дать ответ после перерыва", - заявил "Интерфаксу" полпред ДНР Денис Пушилин. http://www.interfax.ru/world/423235
Кстати, бабушка моя почему-то слово "Дебальцево" не выговаривала, у неё получалось "Гибальцево". О том, что на въезде в город первую букву у города всегда срубали, я уж и не говорю. Местные жители, заметьте.