TotalSynthesisofaNewFluorescentProbefor.doc

精品論文推薦total synthesis of a new fluorescent probe forthe application to intracellular calcium ionshe huaizhen, lei lei, cheng zhao, li jianli, shi zhen *key laboratory of synthetic and natural functional molecule chemistry, ministry ofeducation, department of chemistry, northwest university，xian（710069）e-mail: abstractin this study, a new kind of calcium-selective fluorescent probe fluo-3clmm-oh was synthesized. the structure of reaction products were characterized by ir and 1h nmr spectra. the molecular weights were measured on maldi-tof ms. methylation of the crude product provided aneffectivemethodforseparation.thefluorescenceemissionwavelengthmaximaoffluo-3clmm-oh were also researched.keywords: calcium ions; fluorescent probe; methylation; fluorescence emission1. intr odu c ti on calcium plays a critical role in a broad variety of biological systems. numerous functions of all types of cells are regulated by ca2+ to a greater or lesser degree,1,2 such as calciums role in the beginning of life and the act of fertilization. furthermore, it was shown that ca2+ acted as a universal second messenger and ca2+-binding proteins existed in a variety of cells, which facilitated analysis of the potential functions of ca2+ in a variety of cells.3-5 because of the importance of ca2+ in biology, numerous techniques and methods for investigating the mechanisms of cellular and subcellular ca2+ activity have been developed. among numerousmethods, those based on fluorescence enjoy the benefits of high sensitivity, convenience of dynamic studies and even provide the possibility to visualize the ca2+ distribution inside a cell.6 they are highly sensitive and offer imaging by fluorescent microscopy in an easier and less cell damaging way than other methods (e.g., ion-selective electrodes, nmr techniques).7-10 despite of intensive research efforts, only a few fluorescent indicators are commercially available.11,12among them fluo-3 is a visible-wavelength calcium ions indicator which is convenient for confocal laser scanning microscope (clsm) and flow cytometry.13 however, fluo-3 also suffers some drawbacks.14 its somewhat low affinity to ca2+ does not allow fluo-3 to apply for the observation of small variations in ca2+ concentration. the fluorescence signal is also not satisfactory.here we report the design and synthesis of an improved visible-wavelength calcium-selective fluorescent probe fluo-3mm-oh. it has in common the principle of covalently linking an organic fluorophore to a ca2+-chelating moiety, mostly to 1,2 - bis - ( 2-amino-phenoxy ) ethane - n, n, n, n tetraacetic acid (bapta). introduction of three methyl groups into the ca2+-specific binding site of bapta may increase the electron density and thus improves the affinity of the probe towards a calcium ion. the synthetic route of the compounds is mentioned in scheme 1. methylation of the crude fluo-3clmm-oh, whichreduced the polarity of the product provided an effective method for separation.-4-h3cno2ohah3cno2oclbh3cno2ooch3no2clch3 c12ch3ch3ch3 ch3ch3oo o och3ch3 o o o och3ch3o o o onh2o ocldch3n no o ooch3 en no o oho ch3h3cnh2h3ccl h3c clch3o ch3345ch3 ch3ho o o ohch3 oo o och3ho o o ohho nn oh o oho n n oo o ofoch3 gn no o oo o o o ch3 ho ch3h3c clch3h3c clch3h3c clch3ho o oho o oho o ofluo-3clmm-ohfluo-3clmm-oh esterfluo-3clmm-ohscheme 1: the synthesis rout of fluo-3clmm-oh: (a) brch2ch2cl, dmf, k2co3, 120c, 5 h, 95 % yield. (b) 3, 5 dimethly - 2- nitro- 4- chlorophenol , dmf, k2co3, 150 c, 4 h,92 % yield. (c) fe, hcl, 4 h, 82 % yield. (d) brch2cooch3, i-pr2net, mecn, rt, 35 h, 86 %yield. (e) pocl3, dmf, 45 c,12 h, 82 % yield. (f) resorcinol, meso3h, n2, 50-60 c, o2,80-90 c. (g) anhydrous methanol, 12-24h, dmf, 24-36 h, 80 c. (h) methanol / water, lioh, rt, 12-24h.2. expe rim ental starting materials were commercially available unless otherwise noted. all reagents employed were purchased from xian chemical reagent corp. the melting points were measured on xt-4 instrument without correction. 1h nmr spectra were recorded at 400 mhz in cdcl3 with tms as an internal reference. the ir spectra were recorded on a shimadzu ft-ir 440 spectrometer in 4000-500 cm-1 range. the molecular weights were measured on a matrix assisted laser desorption / ionization time of flight mass spectrometry (maldi-tof ms), using cyano-4-hydroxycinnamic acid (chca) as a reference frame. the synthetic procedure was controlled by thin-layer chromatography (tlc) on precoated silica gel (gf-254). the products were purified by recrystallization or column chromatography silica gel (53 m75 m).as scheme 1 described, the synthesis of compound 1-5 was similar to the previous method 15 with a slight modification. in previous method, compound 3 was obtained through hydrogenating. the reaction was exothermic and not easy to control. in this study, iron powder was used as the reducing agent obtaining a satisfying yield.2.1 preparation of crude f l uo-3clm m -o ha mixture of compound 5 (6.36 g, 10 mmol) and resorcinol (2.20 g, 20 mmol) was added into methanesulfonic acid (20 ml) at room temperature. the mixture was stirred at 50-60 c under dry nitrogen for 10 min, then heated at 80-90 c under oxygen atmosphere for 3-6 h and poured slowly into water (150 ml). the precipitate was filtered off and washed with water. thecrude product was dried in air (2-3 days), and used directly for the next step without further purification.2.2 methylation of f l uo-3clmm-oh the crude acid mixture was dissolved in anhydrous methanol (50-100ml) with the addition of 0.2-0.3 ml of concentrated sulfuric acid. the solution was refluxed for 12-24h then poured into cool water. the crude solid was dried and purified on a silica gel column using 40:1 chloroform/methanol for loading and running the column. 1h nmr (cdcl3, 400 mhz) 7.221 (s, 1h), 7.076 (s, 1h), 6.788-6.763 (m, 2h), 6.906-6.652 (m, 2h), 6.439 (s, 1h), 6.287 (s,1h), 6.114 (s, 1h), 4.322-4.307 (t, 2h), 4.246-4.211 (t, 2h), 4.005 (s, 4h), 3.857 (s, 4h), 3.426 (s, 6h), 3.278 (s, 6h), 2.533 (s, 3h), 2.506 (s, 3h), 2.332 (s, 3h). ir (kbr, , cm-1) 3419.87,2949.53, 1744.23, 1599.22, 1458.96, 1203.81,843.32. maldi/tof ms (m/z): calcd forc42h43cln2o13 819.25, found 8 hydrolysis the solution of purified methyl ester (1.23 g, 1.5 mmol) in methanol (20 ml) / water ( 10 ml) was added lioh pellets ( 1.3 g, 201.6 m mol) slowly and stirred at room temperature for12-24 h. acidification to ph 2 using concentrated hydrochloric acid gave purefluo-3clmm-oh.in addition, the fluorescence emission wavelength maximum of the suggested fluorescein-based calcium indicator is 511 nm, which is near to its predecessor fluo-3.16 thus, fluorescence microscopy applications, omega bandpass filter sets xf104 or xf23 and chroma sets 41028 or 31001 can be used for detection of fluo-3clmm-oh as well.figure 1: the fluorescence emission of fluo-3clmm-oh tablesin summary, for the purpose of developing an improved visible-wavelength calcium-selective fluorescent probe, we have designed and synthesized fluo-3clmm-oh. through methylating the crud acid, we obtained an effective method for separation. further research on the fluorecence properties of the new compound is under studied, and the result will be reported in the near future.ac k n ow le dgm ents this work was support by the national natural science foundation of china (20872117), the specialized research fund for the doctoral program of higher education (20050697003) and the science and technology program foundation of xi an city (yf07070).references1 tsien, r.y. (1983) intracellular measurements of ion activities. ann. rev. biophys. bioeng 12: 101-106. 2 dang, f.f., lei, k.w. & liu, w.s. (2008) a new highly selective fluorescent silver probe. j. fluorese18:149-153.3berridge, m.j. (1993) inositol triphosphate and calcium signalling. nature 361: 315-325.4stricker, s.a., centonze, v.e., paddock, s.w. & schatten, g. (1992) confocal microscopy of fertilization-induced calcium dynamics in sea urchin eggs. dev. biol 149: 370-380.5whitaker, m. & patel, r. (1990) calcium and cell cycle control. development 108: 525-542.6grynkiewicz, g., poenie, g. m. & tsien, r.y. (1985) a new generation of ca2+ indicators withgreatlyimproved fluorescence properties. j. biol. chem 260: 3440-3450.7ridgeway, e.b. & ashley, c.c. (1967) calcium transients in single muscle fibers. biophys. res. commun 29: 229-234.8tsien, r.y. (1981) a non-disruptive technique for loading calcium buffers and indicator into cells.nature 290: 527-528.9tsien, r.y., pozzan, t. & rink, t.j. (1982) calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. j. cell. bio l94: 325-334. 10 komatsu, h., iwasawa, n., citterio, d., suzuki, y. & kubota, t. (2004) design and synthesis of highly sensitive and selective fluorescein-derived magnesi