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pH-sensitive fluorescent deoxyuridines labeled with 2-aminofluorene derivatives
Tetrahedron 72 (2016) 5595e5601
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pH-sensitive fluorescent deoxyuridines labeled with 2-aminofluorene derivatives Ji Won Lee a, Ye-seul Son a, Jung-Yean Lee a, Mi Hyun Kim b, Sang-Keun Woo b, Kyo Chul Lee b, Yong Jin Lee b, Gil Tae Hwang a, * a b
Department of Chemistry, Kyungpook National University, Daegu 41566, Republic of Korea Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences (KIRAMS), Seoul 01812, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 June 2016 Received in revised form 16 July 2016 Accepted 19 July 2016 Available online 20 July 2016
Two fluorescent 20 -deoxyuridines, UAF and UDAF, labeled with 2-aminofluorene and 2-dimethylaminofluorene units, respectively, and having values of pKa of 4.27 and 4.66, respectively, display ‘turn-on’ emission responses in acidic solutions. They can also penetrate into HeLa cell membranes, where they exhibit their strong fluorescence under acidic conditions. Ó 2016 Elsevier Ltd. All rights reserved.
Keywords: Nucleosides Fluorescence Fluorescent probes Deoxyuridine Fluorene
1. Introduction The proton is one of the most important targets in physiological and pathological processes because intracellular pH plays a critical role in many cellular, enzymatic, and tissue activities, including cell growth and apoptosis, ion transport, endocytosis, multidrug resistance, and enzyme activity.1 In particular, abnormal values of intracellular pH are found in such diseases as ischemic stroke,2 cystic fibrosis,3 subarachnoid hemorrhage,4 epilepsy,5 Parkinson’s disease,6 Alzheimer’s disease7 and cancer.8 Therefore, monitoring the pH of living cells in real time remains an important challenge. Fluorescent pH probes offer several attractive features, including high sensitivity, short response time, real-time monitoring and nondestructive identification; these features set such probes apart from other pH measurement methods using, for example, electrodes,9 semiconductor sensors10 or nuclear magnetic resonance (NMR) spectroscopy.9a,11 In particular, fluorescent aromatic amines are useful as pH-responsive probes because they can display photo-induced intramolecular charge transfer (ICT), in which an electron migrates from the amino group to the aromatic ring upon light absorption in their free-base forms,12 whereas protonation of their amino groups terminates their electron-donating
* Corresponding author. Fax: þ82 53 950 6330; e-mail address: [email protected] (G.T. Hwang). http://dx.doi.org/10.1016/j.tet.2016.07.049 0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.
ability. In other words, ICT is influenced in different ways by the pH. Thus, the pH-dependence of the absorption and emission behavior of aromatic amines is of great interest and has been studied to investigate the protonation/deprotonation reactions of aromatic amines in their ground and excited states.1,12d Fluorescent nucleosides bearing aromatic amino moieties are especially attractive because, as nucleoside derivatives, they can be taken up into cells through the action of either concentrative nucleoside transporters or equilibrative nucleoside transporters;13 in the absence of a nucleoside unit, microinjection14 or carriermediated endocytosis15 would have to be used to import fluorescent compounds into cells, and these approaches tend to perturb the physiology of the cell resting state. Although several pHsensitive fluorescent nucleosides have been described,16 there have been few reports of deoxyuridine-bearing aromatic amines as ICT molecules and fluorophores.16b,c Because deoxyuridine labeled with a fluorophore is a nucleoside that can act as a microenvironment-sensitive probe,17 its labeling with aromatic amines would affect its absorption and emission maxima and lead to protonation/ deprotonation-induced on/off fluorescent switching that could be used in aqueous solution to sense pH. Herein, we report two efficient pH-responsive fluorescent probes comprising deoxyuridine derivatives bearing 2aminofluorene (AF) and 2-dimethylaminofluorene (DAF) units, designated UAF and UDAF, respectively.
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2. Results and discussion Scheme 1 presents the synthetic strategy we used to prepare the fluorescent nucleosides UAF and UDAF. 2-Ethynyl-7-trifluoroacetylaminofluorene (3) was prepared through palladium-catalyzed Sonogashira coupling of 118 with TMS-acetylene followed by deprotection with TBAF. Deacetylation of 3 with K2CO3, followed by reductive amination with formaldehyde, yielded compound 5. Subsequent Sonogashira couplings of 20 -deoxy-5-iodouridine (6) with the acetylene derivatives 3 and 5 afforded the fluorescent nucleosides 7 and UDAF, respectively. We obtained UAF through deprotection of 7 with NH4OH. We employed a previously reported procedure to synthesize UFL, bearing the fluorene (FL) unit as a reference probe.17d
acidic conditions. The most remarkable feature of the absorption spectra of DAF and UDAF was that their primary bands grew dramatically upon acidification relative to those of AF and UAF, suggesting that the protonated form containing a DAF moiety absorbed more light at a given wavelength than the protonated form containing an AF moiety. We then examined the fluorescence of all fluorophores in aqueous solutions having various pH values upon excitation at wavelengths near their isosbestic points (Fig. 1). We observed no consistency in the fluorescence intensity of FL upon changing the pH (Fig. 1a), whereas the signals of UFL were relatively redshifted and quenched upon acidification (Fig. 1b). This behavior may have originated from the mesomeric effect and weak acidic properties of the uracil base; N1 of uracil could act as a weak
Scheme 1. Synthesis of UAF and UDAF.
First, we recorded the absorption spectra (see Supplementary data, Fig. S1 and Tables S1eS3) of FL, AF, and DAF (i.e., free fluorene derivatives, not coupled to deoxyuridine units) in 10 mM phosphate buffer at pH 3.10e9.31 at constant ionic strength (0.16 M NaCl). We observed no significant changes in the absorption spectrum of FL in response to the pH change. In contrast, lowering the pH of the solution increased the intensity of the primary absorption band of AF and DAF near 260 nm, originating from the protonated FL moiety, while decreasing the intensity of the secondary band (at approximately 280 nm), attributable to the ICT transition arising from mixing of the p/p* transition of the aromatic ring with the lone pair transition from the amino moiety, with isosbestic points at 270 and 290 nm, respectively. Next, we recorded the absorption spectra of the fluorescent nucleosides containing each FL derivative (UFL, UAF, and UDAF) in the same buffer solution at pH 3.10e9.31 (see Supplementary data, Fig. S2 and Tables S4eS6). Again, we observed no significant changes in the absorption spectrum of UFL, whereas the intensities of the primary bands of UAF and UDAF (approximately 325 and 328 nm, respectively) increased as the intensities of the secondary bands (approximately 340 and 350 nm, respectively) decreased, with isosbestic points at 330 and 350 nm, respectively. These findings are consistent with the protonated amino moieties lacking the electron lone pair necessary for the ICT transition; thus, the absorption spectra of UAF and UDAF were similar to that of UFL under
electron-releasing moiety to the fluorophore through a double bond and triple bond, and this mesomeric effect of UFL was more pronounced under basic conditions because the uracil moiety became a better electron donor by partial deprotonation of its 3NH unit (pKa¼9.5).19 Under acidic conditions, however, the electron releasing properties of a uracil moiety is decreased, which might induce red shifts and decrease in emission intensity. Fig. 1c and e shows the fluorescence spectra of AF and DAF in aqueous solutions having various pH values upon excitation at their isosbestic points (270 and 290 nm, respectively). Generally, lowering the pH of the solution of AF or DAF decreased the intensity of its emission band (approximately 375 and 390 nm, respectively), originating from an ICT-excited state, and increased the intensity of a new emission band (approximately 310 nm), assigned to fluorescence from the excited acidic form of the fluorophore. Notably, upon acidification, where was a difference in the rate of increase in emission intensity of the protonated form of the fluorophore between AF and DAF. For DAF, the emission intensity of the excited acidic form increased at a faster rate than the rate of decrease in emission intensity at the ICT-excited state. This phenomenon originated from the significant increase in absorbance properties of protonated DAF under acidic conditions. Therefore, the fluorescence intensity of DAF increased significantly upon acidification relative that of AF.
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Fig. 1. Emission spectra of (a) FL (excitation wavelength¼260 nm), (b) UFL (330 nm), (c) AF (270 nm), (d) UAF (330 nm), (e) DAF (290 nm) and (f) UDAF (350 nm) at various pH values in 10 mM phosphate buffer (0.16 M NaCl) at 25 C. The concentration of each compound was 2.5 mM. Arrows indicate the direction of acidification.
Upon excitation of UAF and UDAF at their isosbestic points (330 and 350 nm, respectively), emission bands from the non-ICT- and ICT-excited states were found to emerge (Fig. 1d and f). Interestingly, when the pH was decreased, the fluorescence intensities of UDAF were significantly increased relative to those of UAF, in contrast to the behavior of UFL. Because the emission intensities of UAF and UDAF were influenced by their protonated forms upon acidification, the dramatically increased emission intensity of UDAF was assumed to have originated from the relatively sharp increase in the emission intensity of the excited acidic form of DAF. On the other hand, the fluorescence of UAF was not greatly changed upon acidification, consistent with the results observed for AF. Notably, the differences in the fluorescence of each nucleoside under illumination with a transilluminator were readily discernable by the naked eye (Fig. 2a). Plots of the fluorescence intensities of each nucleoside with respect to pH were fitted to the HendersoneHasselbalch equation, giving sigmoidal curves yielding their excited-state pKa values (Fig. 2b).20 Our calculated pKa value for UFL was 7.92, somewhat near the value reported for uridine (pKa¼9.5).19 Therefore, UFL could be deprotonated at its N3 center in alkaline solution, increasing its emission intensity. On the other hand, the nucleosides UAF and UDAF exhibited increased emission intensities under acidic conditions, with pKa values of 4.27 and 4.66, respectively, reflecting the pKa values of their fluorophore units (AF: 4.56; DAF: 4.97; see Supplementary data, Fig. S3). Since the reversible fluorescence responses of pH probes are essential for real-time monitoring reversible pH changes in living organs, we examined whether UAF and UDAF were capable of monitoring reversibly pH changes upon alternate addition of acid and base solutions (Fig. 3). When 2.5 mM of UAF or UDAF in 10 mM phosphate buffer was subjected to pH cycling between pH 3.5 and 7.3 by addition of acid and base to the solution, pH response of UAF and UDAF was highly reversible over at least 10 cycles. The selectivity of UAF and UDAF for protons over the other potential ions was also determined in 10 mM phosphate buffer at pH 3.5 and 7.3. As shown in Fig. 4, UAF and UDAF did not exhibit observable emission change at both pH conditions in the presence of cations, such as Ca2þ, Cd2þ, Co2þ, Cu2þ, Kþ, Mg2þ, Mn2þ, Naþ, and
Ni2þ, as well as amino acids, such as Ala, Cys, Glu, Gly, His, Met, Phe, Pro, and Val. These results indicate that the fluorescence response of UAF and UDAF was unaffected in the presence of background ions. The high reversibility and selectivity of UAF and UDAF indicate that these probes allow their potential application in real-time pH monitoring. To determine UAF and UDAF have potential to be used as probes for investigating intracellular pH, an MTT assay of UAF and UDAF in HeLa cells was first evaluated. In Fig. 5, the MTT assay showed no significant cytotoxic response. Event at high concentrations of 100 mM, the cell viability remained more than 90% with UAF and UDAF. Next, we obtained fluorescence images of HeLa cells treated with UAF and UDAF to examine whether these pH-sensitive nucleosides would display their increased fluorescence intensities under acidic conditions within these cells (Fig. 6). Both nucleosides exhibited relatively strong green emission in the cytosol at pH 4.2, but very weak fluorescence when the cells were treated with the nucleosides at pH 7.3. UDAF displayed brighter emissions than UAF at both pH values. These results revealed that UAF and UDAF both penetrated into the cell membranes and exhibited similar emission behaviors in aqueous solutions and HeLa cells. 3. Conclusion In summary, we have investigated the photophysical properties of the fluorescent 20 -deoxyuridine derivatives UFL (containing a fluorene moiety), UAF (containing a 2-aminofluorene) and UDAF (containing a 2-dimethylaminofluorene) in buffer solutions having various pH values. For UFL, deprotonation of the 3-NH unit (pKa¼7.92) in basic solution led to an enhanced, blue-shifted emission. In contrast, the emission intensities of UAF and UDAF (pKa¼4.27 and 4.66, respectively) were enhanced upon acidification. In particular, UDAF exhibited distinctive fluorescence under acidic conditions due to the highly emissive properties of protonated DAF. Furthermore, UAF and UDAF could penetrate into HeLa cells, exhibiting strong emissions in the acidic cytosol. These pHsensitive nucleosides have the potential to be used as probes for investigating intracellular pH. A deeper understanding of how the
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Fig. 4. Emission spectra of UAF and UDAF (2.5 mM) to common ions and amino acids in acidic buffer solutions with pH¼3.5 (a and c) and neutral buffer solutions with pH¼7.3 (b and d). Analytes used: 0.5 mM for Ca2þ, Mg2þ, 0.3 mM for Cd2þ, Cu2þ, Co2þ, Mn2þ, Ni2þ, 100 mM for Kþ, Naþ, 0.1 mM for amino acids. lex¼330 nm for UAF and 350 nm for UDAF.
Fig. 2. (a) Fluorescence of solutions of UFL, UAF, and UDAF (2.5 mm) in 10 mM phosphate buffer (0.16 M NaCl) at 25 C under a UV transilluminator (312þ365 nm). (b) Normalized emission intensities of UFL (intensity normalized at 476 nm), UAF (459 nm), and UDAF (455 nm) plotted with respect to pH (R2: 0.9770 for UFL; 0.9911 for UAF; and 0.9957 for UDAF). Each point represents three independent measurements; the solid lines are calculated as the best-fit pH titration curves. Fig. 5. Percentages of HeLa cell viabilities assayed by the MTT method.
Fig. 3. Reversible fluoresce response of (a) UAF and (b) UDAF (2.5 mM) to cyclic pH changes between 3.5 and 7.3. Fluorescence intensity was measured at 435 nm for UAF (lex¼330 nm) and 455 for UDAF (lex¼350 nm).
pKa relates to the fluorophore structure could facilitate the design of ideal pH-responsive fluorescent nucleosides. We are currently investigating these concepts in our laboratory.
4. Experimental section 4.1. General All commercially available chemicals were used without further purification. Solvents were carefully dried and distilled prior to use.
Fig. 6. Microscopy (top) and fluorescence (bottom) images of (a) UAF and (b) UDAF at pH 4.2 and 7.3 in HeLa cells. Scale bar is 500 mm.
All reactions were performed in dry glassware under Ar atmospheres. 1 and UFL were synthesized according to procedures reported in the literature.17d,18 Analytical thin layer chromatography (TLC) was performed using Merck 60 F254 silica gel plates; column chromatography was performed using Merck 60 silica gel (230e400 mesh). Melting points were determined using an Electrothermal IA 9000 series melting point apparatus and are uncorrected. Infrared (IR) spectra were recorded using a JASCO FT/IR4100 spectrometer. 1H, 13C, and 19F NMR spectra were recorded
J.W. Lee et al. / Tetrahedron 72 (2016) 5595e5601
using a Bruker NMR spectrometer (AVANCE digital 400 MHz). Data are reported as follows: chemical shifts in ppm (d), multiplicity (s¼singlet, d¼doublet, t¼triplet, q¼quartet, dd¼doublet of doublets, dt¼doublet of triplets, m¼multiplet, br s¼broad singlet), integration, coupling constant (Hz). High-resolution fast atom bombardment (FAB) mass spectra were recorded using a JEOL JMS700 mass spectrometer at the Daegu Center of KBSI, Korea.
4.2. Synthetic procedures 4.2.1. 2,2,2-Trifluoro-N-[7-(trimethylsilyl)ethynyl-9H-fluoren-2-yl] acetamide (2). A solution of 118 (837 mg, 2.35 mmol), (PPh3)2PdCl2 (165 mg, 0.235 mmol) and CuI (44.8 mg, 0.235 mmol) in DMF (35 mL) and Et3N (12 mL) was degassed with argon. Trimethylsilylacetylene (996 mL, 7.05 mmol) was added at 50 C and then the mixture stirred for 4 h. After evaporation of the solvent in vacuo, the residue was taken up into CH2Cl2. The organic phase was washed with water, dried, and concentrated. The residue was subjected to column chromatography (SiO2; hexane/EtOAc, 10:1), yielding a solid (670 mg, 76%): mp 183e185 C; IR (film): n 3839, 3743, 3616, 3459, 3317, 3036, 2918, 2854, 2148, 1701, 1594, 1541, 1463, 1338, 1242, 1164, 928, 820, 756, 701, 653 cm1; 1H NMR (400 MHz, CDCl3): d 7.94 (br s, 1H; NH), 7.90 (d, J¼1.2 Hz, 1H; H-1), 7.75 (d, J¼8.2 Hz, 1H; H-3), 7.67 (d, J¼7.9 Hz, 1H; H-4), 7.65 (d, J¼0.52 Hz, 1H; H-8), 7.50 (dt, J¼7.9, 0.68 Hz, 1H; H-5), 7.46 (dd, J¼8.2, 2.0 Hz, 1H; H-6), 3.89 (s, 2H; H-9), 0.27 (s, 9H; SiCH3); 13C NMR (100 MHz, CDCl3): d 154.9 (q, 2JCF¼37 Hz), 145.1, 143.2, 141.1, 139.5, 134.2, 131.2, 128.7, 121.6, 120.9, 119.8, 119.6, 117.6, 115.9 (d, 1 JCF¼289 Hz), 105.7, 94.6, 36.9, 29.9, 0.2; HRMS-EI (m/z): [M]þ calcd for C20H18F3NOSi, 373.1110; found, 373.1108. 4.2.2. N-(7-Ethynyl-9H-fluoren-2-yl)-2,2,2-trifluoroacetamide (3). Tetra-n-butylammonium fluoride (2.69 mL, 2.69 mmol) was added to a solution of 2 (670 mg, 1.79 mmol) in THF (6.0 mL) and then the mixture was stirred at room temperature for 1.5 h. After evaporation of the solvent in vacuo, the residue was partitioned between with EtOAc and water. The organic phase was washed with water, dried, and concentrated. The residue was purified through column chromatography (SiO2; hexane/EtOAc, 6:1) to give a solid (385 mg, 88%): mp>120 C dec; IR (film): n 3742, 3283, 2920, 2854, 1699, 1605, 1542, 1465, 1415, 1328, 1282, 1167, 915, 860, 816, 699, 649 cm1; 1H NMR (400 MHz, CDCl3): d 8.04 (br s, 1H; NH), 7.91 (d, J¼1.0 Hz, 1H; H-1), 7.76 (d, J¼8.2 Hz, 1H; H-3), 7.70 (d, J¼7.9 Hz, 1H; H-4), 7.66 (s, 1H; H-8), 7.53 (dd, J¼7.8, 0.62 Hz, 1H; H5), 7.48 (dd, J¼8.2, 2.0 Hz, 1H; H-6), 3.91 (s, 2H; H-9), 3.13 (s, 1H; CCH); 13C NMR (100 MHz, CDCl3): d 145.1, 143.2, 141.5, 139.4, 134.3, 131.4, 128.9, 121.0, 120.6, 119.9, 119.6, 117.6, 84.3, 37.0, 29.9; 19F NMR (471 MHz, CDCl3) d 75.6; HRMS-EI (m/z): [M]þ calcd for C17H10F3NO, 301.0714; found, 301.0712. 4.2.3. 7-Ethynyl-9H-fluoren-2-ylamine (4). K2CO3 (2.00 g, 14.4 mmol) was added to a solution of 3 (435 mg, 1.44 mmol) in MeOH (54.8 mL) and H2O (3.3 mL), and then the mixture was heated under reflux for 4 h. The solvent was evaporated and then the residue was partitioned between 1 M NaOH and CH2Cl2. The organic phase was washed with water, dried, and concentrated. The residue was purified through column chromatography (SiO2; hexane/EtOAc, 2:1) to give a solid (255 mg, 86%): mp 116 C; IR (film): n 3840, 3743, 3617, 3398, 3282, 3214, 3015, 2917, 2848, 2363, 2090, 1878, 1751, 1701, 1598, 1454, 1405, 1339, 1265, 1202, 1120, 937, 815, 748, 652, 602 cm1; 1H NMR (400 MHz, CDCl3): d 7.58 (d, J¼0.44 Hz, 1H; H-1), 7.56 (d, J¼3.3 Hz, 1H; H-3), 7.54 (d, J¼3.5 Hz, 1H; H-4), 7.46 (dt, J¼7.8, 0.64 Hz, 1H; H-5), 6.87e6.86 (m, 1H; H-8), 6.71 (dd, J¼8.1, 2.2 Hz, 1H; H-6), 3.79 (s, 2H; H-9), 3.08 (s, 1H; CCH), 1.56 (s, 2H; NH2); 13C NMR (101 MHz, CDCl3): d 146.5, 145.8, 143.0, 142.3, 132.3,
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131.1, 128.5, 121.3, 118.5, 118.3, 114.2, 111.7, 84.9, 76.5, 36.7; HRMS-EI (m/z): [M]þ calcd for C15H11N, 205.0891; found, 205.0893. 4.2.4. (7-Ethynyl-9H-fluoren-2-yl)-N,N-dimethylamine (5). Sodium cyanoborohydride (308 mg, 4.90 mmol) was added to a solution of 4 (205 mg, 0.980 mmol) and paraformaldehyde (351 mg, 11.7 mmol) in glacial AcOH (8.9 mL). After stirring at room temperature under N2 for 18 h, the reaction mixture was poured into ice-water. The precipitate was filtered off, washed with water and 1 M NaOH, and then partitioned between EtOAc and water. The organic phase was dried and concentrated. The residue was purified through column chromatography (SiO2; hexane/EtOAc, 2:1) to give a solid (195 mg, 85%): mp>184 C dec; IR (film): n 3837, 3744, 3677, 3648, 3617, 3291, 3028, 2918, 2850, 2801, 2365, 2095, 1741, 1676, 1605, 1566, 1495, 1463, 1431, 1350, 1295, 1263, 1218, 1182, 1095, 1061, 948, 877, 805, 702, 657, 586 cm1; 1H NMR (400 MHz, CDCl3): d 7.62 (d, J¼8.5 Hz, 1H; H-3), 7.57 (s, 1H; H-1), 7.55 (d, J¼7.9 Hz, 1H; H-4), 7.45 (dt, J¼7.8, 0.68 Hz, 1H; H-5), 6.92 (s, 1H; H8), 6.79 (dd, J¼8.4, 1.9 Hz, 1H; H-6), 3.83 (s, 2H; H-9), 3.07 (s, 1H; CCH), 3.02 (s, 6H; NCH3); 13C NMR (100 MHz, CDCl3): d 145.7, 143.3, 142.3, 131.2, 128.5, 121.0, 118.4, 117.9, 111.9, 109.1, 85.0, 41.1, 37.0, 29.9; HRMS-EI (m/z): [M]þ calcd for C17H15N, 233.1204; found, 233.1203. 4 . 2 . 5 . 2 0 - D e o x y - 5 - [ N - ( 7 - e t h y n yl - 9 H - fl u o r e n - 2 - yl ) - 2 , 2 , 2 trifluoroacetamino]uridine (7). (PPh3)2PdCl2 (91.2 mg, 0.132 mmol) and CuI (24.8 mg, 0.132 mmol) were added to a solution of 20 -deoxy-5-iodouridine (466 mg, 1.32 mmol) and 3 (436 mg, 1.45 mmol) in Et3N (5.7 mL) and DMF (17.3 mL). After Ar had been bubbled through the solution for 2 min, the mixture was subjected to 10 pump/purge cycles and then it was stirred at 50 C for 4 h. After evaporation of solvent in vacuo, the residue was subjected to column chromatography (SiO2; CH2Cl2/MeOH, 40:1) to yield a solid (460 mg, 66%): mp>235 C dec; IR (film): n 3842, 3743, 3672, 3614, 3228, 3101, 2920, 2843, 2361, 1701, 1666, 1553, 1462, 1417, 1261, 1189, 1147, 1099, 1060, 1021, 805, 697 cm1; 1H NMR (400 MHz, DMSO-d6): d 11.70 (br s, 1H; TFA-H), 11.43 (br s, 1H; NeH), 8.41 (s, 1H; H-6), 7.97e7.94 (m, 2H: fluorene-H), 7.90 (d, J¼7.9 Hz, 1H; fluorene-H), 7.69e7.66 (m, 2H; fluorene-H), 7.49 (d, J¼7.9 Hz, 2H; fluorene-H), 6.14 (t, J¼6.4 Hz, 1H; H-10 ), 5.29 (d, J¼4.3 Hz, 1H; OH30 ), 5.22 (t, J¼4.8 Hz, 1H; OH-50 ), 4.28e4.25 (m, 1H; H-30 ), 3.98 (s, 2H; ArCH2), 3.82 (q, J¼3.2 Hz, 1H; H-40 ), 3.70e3.57 (m, 2H; H-50 ), 2.23e2.12 (m, 2H; H-20 ); 13C NMR (100 MHz, DMSO-d6): d 161.5, 154.5 (d, 1JCF¼37 Hz), 149.5, 144.4, 143.7, 143.5, 140.9, 137.9, 135.6, 130.1, 127.8, 120.9, 120.4, 120.2, 120.1, 117.9, 115.9 (d, 2JCF¼289 Hz), 98.4, 92.6, 87.6, 84.9, 82.6, 70.0, 60.9, 36.5; 19F NMR (471 MHz, DMSO-d6) d 73.8; HRMS-FAB (m/z): [MþNa]þ calcd for C26H20F3N3O6Na, 550.1202; found, 550.1205. 4.2.6. 2 0 -Deoxy-5-(7-amino-9H-fluoren-2-ylethynyl)uridine (UAF). Ammonia solution (3.5 mL) was added to 6 (251 mg, 0.476 mmol) in MeOH (1.8 mL) and then the mixture was heated under reflux for 20 h. After evaporation of the solvent in vacuo, the residue was recrystallized (MeOH) to give a solid (145 mg, 71%): mp>207 C dec; IR (film): n 3294, 3160, 3040, 2897, 2823, 2212, 1694, 1457, 1273, 1193, 1095, 990, 816, 689, 609 cm1; 1H NMR (400 MHz, DMSO-d6): d 11.67 (s, 1H; NH), 8.35 (s, 1H; H-6), 7.61 (d, J¼7.9 Hz, 1H; fluorene-H), 7.54 (d, J¼8.2 Hz, 1H; fluorene-H), 7.51 (s, 1H; fluorene-H), 7.36 (dd, J¼7.8, 1.3 Hz, 1H; fluorene-H), 6.77 (s, 1H; fluorene-H), 6.60 (dd, J¼8.2, 1.5 Hz, 1H; fluorene-H), 6.15 (t, J¼6.6 Hz, 1H; H-10 ), 5.39 (br s, 2H; NH2), 5.27 (d, J¼3.5 Hz, 1H; OH30 ), 5.18 (t, J¼4.6 Hz, 1H; OH-50 ), 4.28e4.24 (m, 1H; H-30 ), 3.82 (q, J¼3.2 Hz, 1H; H-40 ), 3.76 (s, 2H; ArCH2), 3.68e3.58 (m, 2H; H-50 ), 2.22e2.17 (m, 2H; H-20 ); 13C NMR (100 MHz, DMSO-d6): d 161.9, 149.8, 145.6, 143.6, 143.2, 142.3, 130.3, 129.3, 127.7, 121.5, 118.4, 118.0, 113.5, 110.6, 99.1, 93.5, 88.0, 85.1, 81.9, 79.5, 70.4, 61.3, 36.4;
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HRMS-FAB (m/z): [MþH]þ calcd for C24H22N3O5, 432.1561; found, 432.1562. 4.2.7. 20 -Deoxy-5-(7-N,N-dimethylamino-9H-fluoren-2-ylethynyl) uridine (UDAF). (PPh3)2PdCl2 (9.90 mg, 0.0141 mmol) and CuI (2.70 mg, 0.0141 mmol) were added to a solution of 20 -deoxy-5iodouridine (49.8 mg, 0.141 mmol) and 5 (49.2 mg, 0.211 mmol) in Et3N (0.6 mL) and DMF (1.8 mL). After Ar had been bubbled through the solution for 2 min, the mixture was subjected to 10 pump/purge cycle and then it was stirred at 50 C for 4 h. After evaporation of the solvent in vacuo, the residue was subjected to column chromatography (SiO2; CH2Cl2/MeOH, 40:1) to yield a solid (30.0 mg, 46%): mp>244 C dec; IR (film): n 3838, 3739, 3614, 3377, 3161, 3046, 2808, 2360, 2206, 1672, 1609, 1502, 1457, 1356, 1275, 1200, 1096, 992, 924, 800, 701, 652, 601 cm1; 1H NMR (400 MHz, DMSO-d6): d 11.68 (s, 1H; NH), 8.36 (s, 1H; H-6), 7.70 (d, J¼3.8 Hz, 1H; fluorene-H), 7.68 (d, J¼3.1 Hz, 1H; fluorene-H), 7.54 (s, 1H; fluorene-H), 7.39 (d, J¼7.9 Hz, 1H; fluorene-H), 6.95 (s, 1H; fluorene-H), 6.77 (dd, J¼8.5, 2.0 Hz, 1H; fluorene-H), 6.14 (t, J¼6.5 Hz, 1H; H-10 ), 5.27 (d, J¼4.2 Hz, 1H; OH-30 ), 5.19 (t, J¼4.8 Hz, 1H; OH-50 ), 4.28e4.27 (m, 1H; H-30 ), 3.84 (s, 2H; ArCH2), 3.82e3.80 (m, 1H; H-40 ), 3.69e3.57 (m, 2H; H-50 ), 2.97 (s, 6H; NCH3), 2.22e2.12 (m, 2H; H-20 ); 13C NMR (100 MHz, DMSO-d6): d 161.9, 150.8, 149.8, 145.6, 143.7, 142.9, 142.5, 130.4, 129.4, 127.8, 121.4, 118.8, 118.3, 111.8, 109.0, 99.0, 93.5, 88.0, 85.2, 82.0, 79.5, 70.4, 61.2, 36.7; HRMS-FAB (m/z): [MþH]þ calcd for C26H26N3O5, 460.1874; found, 460.1870.
bromide (MTT) assay (Sigma) according to the manufacturer’s instruction. 4.7. Fluorescence imaging HeLa human cervical cancer cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; WelGENE Inc., Korea) containing 10% fetal bovine serum (FBS) and 1% antibiotics and were grown in a humidified incubator at 37 C in an atmosphere containing 5% CO2. For experiments, HeLa cells (2105) were plated in six-well culture plates and incubated with DMEM containing 10% FBS at 37 C under humidified conditions with 5% CO2 for 24 h. A solution of nucleoside (UAF or UDAF, 10 mM) in phenol red-free DMEM containing 2% FBS was added to each well, and the HeLa cells were then incubated at 37 C for 18 h. After incubation, the supernatant was removed, and the cells were washed twice with PBS. Phenol redfree DMEM containing 2% FBS (pH adjusted to 4.2 or 7.3) was added. After incubation for 2 h, we recorded the fluorescence images. Acknowledgements This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014R1A1A2056281 and NRF2012R1A1A2044945) and KOSEF by MEST (NRF-2011-0012830). Supplementary data
4.3. UV absorption measurements Ultraviolet (UV) spectra were recorded at various values of pH in 10 mM phosphate buffer (0.16 M NaCl) at 25 C using a Cary 100 UVevis spectrophotometer and 10-mm-path quartz cell, with respect to a pure-solvent reference. All samples were prepared from a stock solution in DMSO to ensure solubility, and hence, all samples contain 0.5% DMSO. 4.4. Fluorescence experiments All samples were prepared as described above for the UV absorption measurements. Fluorescence spectra were recorded using a Cary Eclipse fluorescence spectrophotometer (cell path length: 1 cm; excitation at 260 nm for FL, 270 nm for AF, 290 nm for DAF, 330 nm for UFL, 330 nm for UAF and 350 nm for UDAF). The excitation and emission bandwidth was 1 nm. The fluorescence quantum yields (FF) were determined using solution of pyrene in cyclohexane or fluorene in EtOH as a reference.21 The pKa value was determined by interpolation of a Boltzmann sigmoidal curve between the normalized fluorescence intensity versus pH. 4.5. pH cycling and selectivity experiments The pH cycling and selectivity experiments were performed at room temperature. UAF or UDAF (2.5 mM) was cycled between pH 3.5 and 7.3 by alternate addition of 5.3 mL 5 M HCl and 5.8 mL of 5 M NaOH, respectively. The stock solutions of metal ions and amino acids were prepared from KCl, NaCl, CaCl2, MgSO4, CdSO4$H2O, CuCl2$2H2O, CoCl2$6H2O, MnCl2$4H2O, NiCl2$6H2O, L-Glutamic acid, L-Cystein, L-Histidine, L-Proline, L-Phenylalanine, L-Glycine, LAlanine, L-Valine, L-Methionine. 4.6. MTT assay for the cell cytotoxicity The cytotoxicity measurements were evaluated in a HeLa cell line by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tet.2016.07.049. References and notes 1. (a) Yin, J.; Hu, Y.; Yoon, J. Chem. Soc. Rev. 2015, 44, 4619e4644; (b) Lee, M. H.; Park, N.; Yi, C.; Han, J. H.; Hong, J. H.; Kim, K. P.; Kang, D. H.; Sessler, J. L.; Kang, C.; Kim, J. S. J. Am. Chem. Soc. 2014, 40, 14136e14142; (c) Ni, Y.; Wu, J. Org. Biomol. Chem. 2014, 23, 3774e3791; (d) Han, J.; Burgess, K. Chem. Rev. 2010, 110, 2709e2728; (e) Wang, R.; Yu, C.; Yu, F.; Chen, L. Trends Anal. Chem. 2010, 29, 1004e1013 and references therein. 2. Lam, T. I.; Brennan-Minnella, A. M.; Won, S. J.; Shen, Y.; Hefner, C.; Shi, Y.; Sun, D.; Swanson, R. A. Proc. Natl. Acad. Sci. U.S.A. 2013, 46, E4362eE4368. 3. Coakley, R. J.; Taggart, C.; Canny, G.; Greally, P.; O’Neill, S. J.; McElvaney, N. G. Am. J. Physiol. Lung Cell. Mol. Physiol. 2000, 1, L66eL74. 4. Brooke, N. S.; Ouwerkerk, R.; Adams, C. B.; Radda, G. K.; Ledingham, J. G.; Rajagopalan, B. Proc. Natl. Acad. Sci. U.S.A. 1994, 5, 1903e1907. 5. Andrade, C. S.; Otaduy, M. C.; Valente, K. D.; Park, E. J.; Kanas, A. F.; Silva Filho, M. R.; Tsunemi, M. H.; Leite, C. C. Brain Dev. 2014, 10, 899e906. 6. Cole, N. B.; Dieuliis, D.; Leo, P.; Mitchell, D. C.; Nussbaum, R. L. Exp. Cell Res. 2008, 10, 2076e2089. 7. (a) Reddy, P. H.; Tripathi, R.; Troung, Q.; Tirumala, K.; Reddy, T. P.; Anekonda, V.; Shirendeb, U. P.; Calkins, M. J.; Reddy, A. P.; Mao, P.; Manczak, M. Biochim. Biophys. Acta 2012, 5, 639e649; (b) Harguindey, S.; Reshkin, S. J.; Orive, G.; Arranz, J. L.; Anitua, E. Curr. Alzheimer Res. 2007, 1, 53e65. 8. (a) Wojtkowiak, J. W.; Rothberg, J. M.; Kumar, V.; Schramm, K. J.; Haller, E.; Proemsey, J. B.; Lloyd, M. C.; Sloane, B. F.; Gillies, R. J. Cancer Res. 2012, 16, 3938e3947; (b) Riemann, A.; Schneider, B.; Ihling, A.; Nowak, M.; Sauvant, C.; Thews, O.; Gekle, M. PLoS One 2011, 6 e22445: 1e12. 9. (a) Loiselle, F. B.; Casey, J. R. Methods Mol. Biol. 2010, 637, 311e331; (b) Buck, R. P.; Rondinini, S.; Covington, A. K.; Baucke, F. G. K.; Brett, C. M. A.; Camoes, M. F.; Milton, M. J. T.; Mussini, T.; Naumann, R.; Pratt, K. W.; Spitzer, P.; Wilson, G. S. Pure Appl. Chem. 2002, 74, 2169e2200. 10. Devadhasan, J. P.; Kim, S. Anal. Chim. Acta 2015, 858, 55e59. 11. (a) Thaplyal, P.; Bevilacqua, P. C. Methods Enzymol. 2014, 549, 189e219; (b) Rata, M.; Giles, S. L.; deSouza, N. M.; Leach, M. O.; Payne, G. S. NMR Biomed. 2014, 2, 158e162. 12. (a) Linton, K. E.; Fox, M. A.; P alsson, L.-O.; Bryce, M. R. Chem.dEur. J. 2015, 21, 3997e4007; (b) Karasulu, B.; Thiel, W. J. Phys. Chem. B 2015, 3, 928e943; (c) Chudomel, J. M.; Yang, B.; Barnes, M. D.; Achermann, M.; Mague, J. T.; Lahti, P. M. J. Phys. Chem. A 2011, 30, 8361e8368; (d) Park, K. K.; Park, J. W.; Hamilton, A. D. J. Fluoresc. 2007, 4, 361e369. 13. (a) Guida, L.; Bruzzone, S.; Sturla, L.; Franco, L.; Zocchi, E.; De Flora, A. J. Biol. Chem. 2002, 49, 47097e47105; (b) Moss, A. M.; Endres, C. J.; Ruiz-Garcia, A.; Choi, D. S.; Unadkat, J. D. Mol. Pharmacol. 2012, 9, 2442e2449; (c) Marechal, R.; Mackey, J. R.; Lai, R.; Demetter, P.; Peeters, M.; Polus, M.; Cass, C. E.; Young, J.; Salmon, I.; Deviere, J.; Van Laethem, J. L. Clin. Cancer Res. 2009, 8, 2913e2919.
J.W. Lee et al. / Tetrahedron 72 (2016) 5595e5601 14. Sbriccoli, A.; Carretta, D.; Santarelli, M.; Pinto, F.; Granato, A.; Minciacchi, D. Brain Res. Protoc. 1999, 3, 257e263. 15. Sonawane, N. D.; Verkman, A. S. J. Cell Biol. 2003, 7, 1129e1138. 16. (a) Sinkeldam, R. W.; Hopkins, P. A.; Tor, Y. ChemPhysChem 2012, 14, 3350e3356; (b) Saito, Y.; Miyamoto, S.; Suzuki, A.; Matsumoto, K.; Ishihara, T.; Saito, I. Bioorg. Med. Chem. Lett. 2012, 8, 2753e2756; (c) Riedl, J.; Pohl, R.; Rulisek, L.; Hocek, M. J. Org. Chem. 2012, 2, 1026e1044; (d) Sinkeldam, R. W.; Marcus, P.; Uchenik, D.; Tor, Y. ChemPhysChem 2011, 12, 2260e2265; (e) Sun, K. M.; McLaughlin, C. K.; Lantero, D. R.; Manderville, R. A. J. Am. Chem. Soc. 2007, 7, 1894e1895 and references therein. 17. (a) Lee, J.; Cho, H. Y.; Hwang, G. T. ChemBioChem 2013, 11, 1353e1362; (b) Cho, H. Y.; Woo, S. K.; Hwang, G. T. Molecules 2012, 17, 12061e12071; (c) Ryu, J. H.;
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Heo, J. Y.; Bang, E. K.; Hwang, G. T.; Kim, B. H. Tetrahedron 2012, 68, 72e78; (d) Hwang, G. T.; Seo, Y. J.; Kim, B. H. J. Am. Chem. Soc. 2004, 21, 6528e6529 and references therein. Pan, H.-L.; Fletcher, T. L. J. Med. Chem. 1964, 7, 31e38. (a) Thibaudeau, C.; Plavec, J.; Chattopadhyaya, J. J. Org. Chem. 1996, 61, 266e286; (b) Izatt, R. M.; Christensen, J. J.; Rytting, J. H. Chem. Rev. 1971, 5, 439e481. The Determination of Ionization Constants: a Laboratory Manual, 3rd ed.; Albert, A., Serjeant, E. P., Eds., 3rd ed.; Chapman & Hall: New York, 1984; pp 70e101. Photoluminescence of Solutions; Parker, C. A., Ed.; Elsevier: Amsterdam, 1968; pp 261e268.
Contents lists available at ScienceDirect
Tetrahedron journal homepage: www.elsevier.com/locate/tet
pH-sensitive fluorescent deoxyuridines labeled with 2-aminofluorene derivatives Ji Won Lee a, Ye-seul Son a, Jung-Yean Lee a, Mi Hyun Kim b, Sang-Keun Woo b, Kyo Chul Lee b, Yong Jin Lee b, Gil Tae Hwang a, * a b
Department of Chemistry, Kyungpook National University, Daegu 41566, Republic of Korea Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences (KIRAMS), Seoul 01812, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 June 2016 Received in revised form 16 July 2016 Accepted 19 July 2016 Available online 20 July 2016
Two fluorescent 20 -deoxyuridines, UAF and UDAF, labeled with 2-aminofluorene and 2-dimethylaminofluorene units, respectively, and having values of pKa of 4.27 and 4.66, respectively, display ‘turn-on’ emission responses in acidic solutions. They can also penetrate into HeLa cell membranes, where they exhibit their strong fluorescence under acidic conditions. Ó 2016 Elsevier Ltd. All rights reserved.
Keywords: Nucleosides Fluorescence Fluorescent probes Deoxyuridine Fluorene
1. Introduction The proton is one of the most important targets in physiological and pathological processes because intracellular pH plays a critical role in many cellular, enzymatic, and tissue activities, including cell growth and apoptosis, ion transport, endocytosis, multidrug resistance, and enzyme activity.1 In particular, abnormal values of intracellular pH are found in such diseases as ischemic stroke,2 cystic fibrosis,3 subarachnoid hemorrhage,4 epilepsy,5 Parkinson’s disease,6 Alzheimer’s disease7 and cancer.8 Therefore, monitoring the pH of living cells in real time remains an important challenge. Fluorescent pH probes offer several attractive features, including high sensitivity, short response time, real-time monitoring and nondestructive identification; these features set such probes apart from other pH measurement methods using, for example, electrodes,9 semiconductor sensors10 or nuclear magnetic resonance (NMR) spectroscopy.9a,11 In particular, fluorescent aromatic amines are useful as pH-responsive probes because they can display photo-induced intramolecular charge transfer (ICT), in which an electron migrates from the amino group to the aromatic ring upon light absorption in their free-base forms,12 whereas protonation of their amino groups terminates their electron-donating
* Corresponding author. Fax: þ82 53 950 6330; e-mail address: [email protected] (G.T. Hwang). http://dx.doi.org/10.1016/j.tet.2016.07.049 0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.
ability. In other words, ICT is influenced in different ways by the pH. Thus, the pH-dependence of the absorption and emission behavior of aromatic amines is of great interest and has been studied to investigate the protonation/deprotonation reactions of aromatic amines in their ground and excited states.1,12d Fluorescent nucleosides bearing aromatic amino moieties are especially attractive because, as nucleoside derivatives, they can be taken up into cells through the action of either concentrative nucleoside transporters or equilibrative nucleoside transporters;13 in the absence of a nucleoside unit, microinjection14 or carriermediated endocytosis15 would have to be used to import fluorescent compounds into cells, and these approaches tend to perturb the physiology of the cell resting state. Although several pHsensitive fluorescent nucleosides have been described,16 there have been few reports of deoxyuridine-bearing aromatic amines as ICT molecules and fluorophores.16b,c Because deoxyuridine labeled with a fluorophore is a nucleoside that can act as a microenvironment-sensitive probe,17 its labeling with aromatic amines would affect its absorption and emission maxima and lead to protonation/ deprotonation-induced on/off fluorescent switching that could be used in aqueous solution to sense pH. Herein, we report two efficient pH-responsive fluorescent probes comprising deoxyuridine derivatives bearing 2aminofluorene (AF) and 2-dimethylaminofluorene (DAF) units, designated UAF and UDAF, respectively.
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2. Results and discussion Scheme 1 presents the synthetic strategy we used to prepare the fluorescent nucleosides UAF and UDAF. 2-Ethynyl-7-trifluoroacetylaminofluorene (3) was prepared through palladium-catalyzed Sonogashira coupling of 118 with TMS-acetylene followed by deprotection with TBAF. Deacetylation of 3 with K2CO3, followed by reductive amination with formaldehyde, yielded compound 5. Subsequent Sonogashira couplings of 20 -deoxy-5-iodouridine (6) with the acetylene derivatives 3 and 5 afforded the fluorescent nucleosides 7 and UDAF, respectively. We obtained UAF through deprotection of 7 with NH4OH. We employed a previously reported procedure to synthesize UFL, bearing the fluorene (FL) unit as a reference probe.17d
acidic conditions. The most remarkable feature of the absorption spectra of DAF and UDAF was that their primary bands grew dramatically upon acidification relative to those of AF and UAF, suggesting that the protonated form containing a DAF moiety absorbed more light at a given wavelength than the protonated form containing an AF moiety. We then examined the fluorescence of all fluorophores in aqueous solutions having various pH values upon excitation at wavelengths near their isosbestic points (Fig. 1). We observed no consistency in the fluorescence intensity of FL upon changing the pH (Fig. 1a), whereas the signals of UFL were relatively redshifted and quenched upon acidification (Fig. 1b). This behavior may have originated from the mesomeric effect and weak acidic properties of the uracil base; N1 of uracil could act as a weak
Scheme 1. Synthesis of UAF and UDAF.
First, we recorded the absorption spectra (see Supplementary data, Fig. S1 and Tables S1eS3) of FL, AF, and DAF (i.e., free fluorene derivatives, not coupled to deoxyuridine units) in 10 mM phosphate buffer at pH 3.10e9.31 at constant ionic strength (0.16 M NaCl). We observed no significant changes in the absorption spectrum of FL in response to the pH change. In contrast, lowering the pH of the solution increased the intensity of the primary absorption band of AF and DAF near 260 nm, originating from the protonated FL moiety, while decreasing the intensity of the secondary band (at approximately 280 nm), attributable to the ICT transition arising from mixing of the p/p* transition of the aromatic ring with the lone pair transition from the amino moiety, with isosbestic points at 270 and 290 nm, respectively. Next, we recorded the absorption spectra of the fluorescent nucleosides containing each FL derivative (UFL, UAF, and UDAF) in the same buffer solution at pH 3.10e9.31 (see Supplementary data, Fig. S2 and Tables S4eS6). Again, we observed no significant changes in the absorption spectrum of UFL, whereas the intensities of the primary bands of UAF and UDAF (approximately 325 and 328 nm, respectively) increased as the intensities of the secondary bands (approximately 340 and 350 nm, respectively) decreased, with isosbestic points at 330 and 350 nm, respectively. These findings are consistent with the protonated amino moieties lacking the electron lone pair necessary for the ICT transition; thus, the absorption spectra of UAF and UDAF were similar to that of UFL under
electron-releasing moiety to the fluorophore through a double bond and triple bond, and this mesomeric effect of UFL was more pronounced under basic conditions because the uracil moiety became a better electron donor by partial deprotonation of its 3NH unit (pKa¼9.5).19 Under acidic conditions, however, the electron releasing properties of a uracil moiety is decreased, which might induce red shifts and decrease in emission intensity. Fig. 1c and e shows the fluorescence spectra of AF and DAF in aqueous solutions having various pH values upon excitation at their isosbestic points (270 and 290 nm, respectively). Generally, lowering the pH of the solution of AF or DAF decreased the intensity of its emission band (approximately 375 and 390 nm, respectively), originating from an ICT-excited state, and increased the intensity of a new emission band (approximately 310 nm), assigned to fluorescence from the excited acidic form of the fluorophore. Notably, upon acidification, where was a difference in the rate of increase in emission intensity of the protonated form of the fluorophore between AF and DAF. For DAF, the emission intensity of the excited acidic form increased at a faster rate than the rate of decrease in emission intensity at the ICT-excited state. This phenomenon originated from the significant increase in absorbance properties of protonated DAF under acidic conditions. Therefore, the fluorescence intensity of DAF increased significantly upon acidification relative that of AF.
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Fig. 1. Emission spectra of (a) FL (excitation wavelength¼260 nm), (b) UFL (330 nm), (c) AF (270 nm), (d) UAF (330 nm), (e) DAF (290 nm) and (f) UDAF (350 nm) at various pH values in 10 mM phosphate buffer (0.16 M NaCl) at 25 C. The concentration of each compound was 2.5 mM. Arrows indicate the direction of acidification.
Upon excitation of UAF and UDAF at their isosbestic points (330 and 350 nm, respectively), emission bands from the non-ICT- and ICT-excited states were found to emerge (Fig. 1d and f). Interestingly, when the pH was decreased, the fluorescence intensities of UDAF were significantly increased relative to those of UAF, in contrast to the behavior of UFL. Because the emission intensities of UAF and UDAF were influenced by their protonated forms upon acidification, the dramatically increased emission intensity of UDAF was assumed to have originated from the relatively sharp increase in the emission intensity of the excited acidic form of DAF. On the other hand, the fluorescence of UAF was not greatly changed upon acidification, consistent with the results observed for AF. Notably, the differences in the fluorescence of each nucleoside under illumination with a transilluminator were readily discernable by the naked eye (Fig. 2a). Plots of the fluorescence intensities of each nucleoside with respect to pH were fitted to the HendersoneHasselbalch equation, giving sigmoidal curves yielding their excited-state pKa values (Fig. 2b).20 Our calculated pKa value for UFL was 7.92, somewhat near the value reported for uridine (pKa¼9.5).19 Therefore, UFL could be deprotonated at its N3 center in alkaline solution, increasing its emission intensity. On the other hand, the nucleosides UAF and UDAF exhibited increased emission intensities under acidic conditions, with pKa values of 4.27 and 4.66, respectively, reflecting the pKa values of their fluorophore units (AF: 4.56; DAF: 4.97; see Supplementary data, Fig. S3). Since the reversible fluorescence responses of pH probes are essential for real-time monitoring reversible pH changes in living organs, we examined whether UAF and UDAF were capable of monitoring reversibly pH changes upon alternate addition of acid and base solutions (Fig. 3). When 2.5 mM of UAF or UDAF in 10 mM phosphate buffer was subjected to pH cycling between pH 3.5 and 7.3 by addition of acid and base to the solution, pH response of UAF and UDAF was highly reversible over at least 10 cycles. The selectivity of UAF and UDAF for protons over the other potential ions was also determined in 10 mM phosphate buffer at pH 3.5 and 7.3. As shown in Fig. 4, UAF and UDAF did not exhibit observable emission change at both pH conditions in the presence of cations, such as Ca2þ, Cd2þ, Co2þ, Cu2þ, Kþ, Mg2þ, Mn2þ, Naþ, and
Ni2þ, as well as amino acids, such as Ala, Cys, Glu, Gly, His, Met, Phe, Pro, and Val. These results indicate that the fluorescence response of UAF and UDAF was unaffected in the presence of background ions. The high reversibility and selectivity of UAF and UDAF indicate that these probes allow their potential application in real-time pH monitoring. To determine UAF and UDAF have potential to be used as probes for investigating intracellular pH, an MTT assay of UAF and UDAF in HeLa cells was first evaluated. In Fig. 5, the MTT assay showed no significant cytotoxic response. Event at high concentrations of 100 mM, the cell viability remained more than 90% with UAF and UDAF. Next, we obtained fluorescence images of HeLa cells treated with UAF and UDAF to examine whether these pH-sensitive nucleosides would display their increased fluorescence intensities under acidic conditions within these cells (Fig. 6). Both nucleosides exhibited relatively strong green emission in the cytosol at pH 4.2, but very weak fluorescence when the cells were treated with the nucleosides at pH 7.3. UDAF displayed brighter emissions than UAF at both pH values. These results revealed that UAF and UDAF both penetrated into the cell membranes and exhibited similar emission behaviors in aqueous solutions and HeLa cells. 3. Conclusion In summary, we have investigated the photophysical properties of the fluorescent 20 -deoxyuridine derivatives UFL (containing a fluorene moiety), UAF (containing a 2-aminofluorene) and UDAF (containing a 2-dimethylaminofluorene) in buffer solutions having various pH values. For UFL, deprotonation of the 3-NH unit (pKa¼7.92) in basic solution led to an enhanced, blue-shifted emission. In contrast, the emission intensities of UAF and UDAF (pKa¼4.27 and 4.66, respectively) were enhanced upon acidification. In particular, UDAF exhibited distinctive fluorescence under acidic conditions due to the highly emissive properties of protonated DAF. Furthermore, UAF and UDAF could penetrate into HeLa cells, exhibiting strong emissions in the acidic cytosol. These pHsensitive nucleosides have the potential to be used as probes for investigating intracellular pH. A deeper understanding of how the
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Fig. 4. Emission spectra of UAF and UDAF (2.5 mM) to common ions and amino acids in acidic buffer solutions with pH¼3.5 (a and c) and neutral buffer solutions with pH¼7.3 (b and d). Analytes used: 0.5 mM for Ca2þ, Mg2þ, 0.3 mM for Cd2þ, Cu2þ, Co2þ, Mn2þ, Ni2þ, 100 mM for Kþ, Naþ, 0.1 mM for amino acids. lex¼330 nm for UAF and 350 nm for UDAF.
Fig. 2. (a) Fluorescence of solutions of UFL, UAF, and UDAF (2.5 mm) in 10 mM phosphate buffer (0.16 M NaCl) at 25 C under a UV transilluminator (312þ365 nm). (b) Normalized emission intensities of UFL (intensity normalized at 476 nm), UAF (459 nm), and UDAF (455 nm) plotted with respect to pH (R2: 0.9770 for UFL; 0.9911 for UAF; and 0.9957 for UDAF). Each point represents three independent measurements; the solid lines are calculated as the best-fit pH titration curves. Fig. 5. Percentages of HeLa cell viabilities assayed by the MTT method.
Fig. 3. Reversible fluoresce response of (a) UAF and (b) UDAF (2.5 mM) to cyclic pH changes between 3.5 and 7.3. Fluorescence intensity was measured at 435 nm for UAF (lex¼330 nm) and 455 for UDAF (lex¼350 nm).
pKa relates to the fluorophore structure could facilitate the design of ideal pH-responsive fluorescent nucleosides. We are currently investigating these concepts in our laboratory.
4. Experimental section 4.1. General All commercially available chemicals were used without further purification. Solvents were carefully dried and distilled prior to use.
Fig. 6. Microscopy (top) and fluorescence (bottom) images of (a) UAF and (b) UDAF at pH 4.2 and 7.3 in HeLa cells. Scale bar is 500 mm.
All reactions were performed in dry glassware under Ar atmospheres. 1 and UFL were synthesized according to procedures reported in the literature.17d,18 Analytical thin layer chromatography (TLC) was performed using Merck 60 F254 silica gel plates; column chromatography was performed using Merck 60 silica gel (230e400 mesh). Melting points were determined using an Electrothermal IA 9000 series melting point apparatus and are uncorrected. Infrared (IR) spectra were recorded using a JASCO FT/IR4100 spectrometer. 1H, 13C, and 19F NMR spectra were recorded
J.W. Lee et al. / Tetrahedron 72 (2016) 5595e5601
using a Bruker NMR spectrometer (AVANCE digital 400 MHz). Data are reported as follows: chemical shifts in ppm (d), multiplicity (s¼singlet, d¼doublet, t¼triplet, q¼quartet, dd¼doublet of doublets, dt¼doublet of triplets, m¼multiplet, br s¼broad singlet), integration, coupling constant (Hz). High-resolution fast atom bombardment (FAB) mass spectra were recorded using a JEOL JMS700 mass spectrometer at the Daegu Center of KBSI, Korea.
4.2. Synthetic procedures 4.2.1. 2,2,2-Trifluoro-N-[7-(trimethylsilyl)ethynyl-9H-fluoren-2-yl] acetamide (2). A solution of 118 (837 mg, 2.35 mmol), (PPh3)2PdCl2 (165 mg, 0.235 mmol) and CuI (44.8 mg, 0.235 mmol) in DMF (35 mL) and Et3N (12 mL) was degassed with argon. Trimethylsilylacetylene (996 mL, 7.05 mmol) was added at 50 C and then the mixture stirred for 4 h. After evaporation of the solvent in vacuo, the residue was taken up into CH2Cl2. The organic phase was washed with water, dried, and concentrated. The residue was subjected to column chromatography (SiO2; hexane/EtOAc, 10:1), yielding a solid (670 mg, 76%): mp 183e185 C; IR (film): n 3839, 3743, 3616, 3459, 3317, 3036, 2918, 2854, 2148, 1701, 1594, 1541, 1463, 1338, 1242, 1164, 928, 820, 756, 701, 653 cm1; 1H NMR (400 MHz, CDCl3): d 7.94 (br s, 1H; NH), 7.90 (d, J¼1.2 Hz, 1H; H-1), 7.75 (d, J¼8.2 Hz, 1H; H-3), 7.67 (d, J¼7.9 Hz, 1H; H-4), 7.65 (d, J¼0.52 Hz, 1H; H-8), 7.50 (dt, J¼7.9, 0.68 Hz, 1H; H-5), 7.46 (dd, J¼8.2, 2.0 Hz, 1H; H-6), 3.89 (s, 2H; H-9), 0.27 (s, 9H; SiCH3); 13C NMR (100 MHz, CDCl3): d 154.9 (q, 2JCF¼37 Hz), 145.1, 143.2, 141.1, 139.5, 134.2, 131.2, 128.7, 121.6, 120.9, 119.8, 119.6, 117.6, 115.9 (d, 1 JCF¼289 Hz), 105.7, 94.6, 36.9, 29.9, 0.2; HRMS-EI (m/z): [M]þ calcd for C20H18F3NOSi, 373.1110; found, 373.1108. 4.2.2. N-(7-Ethynyl-9H-fluoren-2-yl)-2,2,2-trifluoroacetamide (3). Tetra-n-butylammonium fluoride (2.69 mL, 2.69 mmol) was added to a solution of 2 (670 mg, 1.79 mmol) in THF (6.0 mL) and then the mixture was stirred at room temperature for 1.5 h. After evaporation of the solvent in vacuo, the residue was partitioned between with EtOAc and water. The organic phase was washed with water, dried, and concentrated. The residue was purified through column chromatography (SiO2; hexane/EtOAc, 6:1) to give a solid (385 mg, 88%): mp>120 C dec; IR (film): n 3742, 3283, 2920, 2854, 1699, 1605, 1542, 1465, 1415, 1328, 1282, 1167, 915, 860, 816, 699, 649 cm1; 1H NMR (400 MHz, CDCl3): d 8.04 (br s, 1H; NH), 7.91 (d, J¼1.0 Hz, 1H; H-1), 7.76 (d, J¼8.2 Hz, 1H; H-3), 7.70 (d, J¼7.9 Hz, 1H; H-4), 7.66 (s, 1H; H-8), 7.53 (dd, J¼7.8, 0.62 Hz, 1H; H5), 7.48 (dd, J¼8.2, 2.0 Hz, 1H; H-6), 3.91 (s, 2H; H-9), 3.13 (s, 1H; CCH); 13C NMR (100 MHz, CDCl3): d 145.1, 143.2, 141.5, 139.4, 134.3, 131.4, 128.9, 121.0, 120.6, 119.9, 119.6, 117.6, 84.3, 37.0, 29.9; 19F NMR (471 MHz, CDCl3) d 75.6; HRMS-EI (m/z): [M]þ calcd for C17H10F3NO, 301.0714; found, 301.0712. 4.2.3. 7-Ethynyl-9H-fluoren-2-ylamine (4). K2CO3 (2.00 g, 14.4 mmol) was added to a solution of 3 (435 mg, 1.44 mmol) in MeOH (54.8 mL) and H2O (3.3 mL), and then the mixture was heated under reflux for 4 h. The solvent was evaporated and then the residue was partitioned between 1 M NaOH and CH2Cl2. The organic phase was washed with water, dried, and concentrated. The residue was purified through column chromatography (SiO2; hexane/EtOAc, 2:1) to give a solid (255 mg, 86%): mp 116 C; IR (film): n 3840, 3743, 3617, 3398, 3282, 3214, 3015, 2917, 2848, 2363, 2090, 1878, 1751, 1701, 1598, 1454, 1405, 1339, 1265, 1202, 1120, 937, 815, 748, 652, 602 cm1; 1H NMR (400 MHz, CDCl3): d 7.58 (d, J¼0.44 Hz, 1H; H-1), 7.56 (d, J¼3.3 Hz, 1H; H-3), 7.54 (d, J¼3.5 Hz, 1H; H-4), 7.46 (dt, J¼7.8, 0.64 Hz, 1H; H-5), 6.87e6.86 (m, 1H; H-8), 6.71 (dd, J¼8.1, 2.2 Hz, 1H; H-6), 3.79 (s, 2H; H-9), 3.08 (s, 1H; CCH), 1.56 (s, 2H; NH2); 13C NMR (101 MHz, CDCl3): d 146.5, 145.8, 143.0, 142.3, 132.3,
5599
131.1, 128.5, 121.3, 118.5, 118.3, 114.2, 111.7, 84.9, 76.5, 36.7; HRMS-EI (m/z): [M]þ calcd for C15H11N, 205.0891; found, 205.0893. 4.2.4. (7-Ethynyl-9H-fluoren-2-yl)-N,N-dimethylamine (5). Sodium cyanoborohydride (308 mg, 4.90 mmol) was added to a solution of 4 (205 mg, 0.980 mmol) and paraformaldehyde (351 mg, 11.7 mmol) in glacial AcOH (8.9 mL). After stirring at room temperature under N2 for 18 h, the reaction mixture was poured into ice-water. The precipitate was filtered off, washed with water and 1 M NaOH, and then partitioned between EtOAc and water. The organic phase was dried and concentrated. The residue was purified through column chromatography (SiO2; hexane/EtOAc, 2:1) to give a solid (195 mg, 85%): mp>184 C dec; IR (film): n 3837, 3744, 3677, 3648, 3617, 3291, 3028, 2918, 2850, 2801, 2365, 2095, 1741, 1676, 1605, 1566, 1495, 1463, 1431, 1350, 1295, 1263, 1218, 1182, 1095, 1061, 948, 877, 805, 702, 657, 586 cm1; 1H NMR (400 MHz, CDCl3): d 7.62 (d, J¼8.5 Hz, 1H; H-3), 7.57 (s, 1H; H-1), 7.55 (d, J¼7.9 Hz, 1H; H-4), 7.45 (dt, J¼7.8, 0.68 Hz, 1H; H-5), 6.92 (s, 1H; H8), 6.79 (dd, J¼8.4, 1.9 Hz, 1H; H-6), 3.83 (s, 2H; H-9), 3.07 (s, 1H; CCH), 3.02 (s, 6H; NCH3); 13C NMR (100 MHz, CDCl3): d 145.7, 143.3, 142.3, 131.2, 128.5, 121.0, 118.4, 117.9, 111.9, 109.1, 85.0, 41.1, 37.0, 29.9; HRMS-EI (m/z): [M]þ calcd for C17H15N, 233.1204; found, 233.1203. 4 . 2 . 5 . 2 0 - D e o x y - 5 - [ N - ( 7 - e t h y n yl - 9 H - fl u o r e n - 2 - yl ) - 2 , 2 , 2 trifluoroacetamino]uridine (7). (PPh3)2PdCl2 (91.2 mg, 0.132 mmol) and CuI (24.8 mg, 0.132 mmol) were added to a solution of 20 -deoxy-5-iodouridine (466 mg, 1.32 mmol) and 3 (436 mg, 1.45 mmol) in Et3N (5.7 mL) and DMF (17.3 mL). After Ar had been bubbled through the solution for 2 min, the mixture was subjected to 10 pump/purge cycles and then it was stirred at 50 C for 4 h. After evaporation of solvent in vacuo, the residue was subjected to column chromatography (SiO2; CH2Cl2/MeOH, 40:1) to yield a solid (460 mg, 66%): mp>235 C dec; IR (film): n 3842, 3743, 3672, 3614, 3228, 3101, 2920, 2843, 2361, 1701, 1666, 1553, 1462, 1417, 1261, 1189, 1147, 1099, 1060, 1021, 805, 697 cm1; 1H NMR (400 MHz, DMSO-d6): d 11.70 (br s, 1H; TFA-H), 11.43 (br s, 1H; NeH), 8.41 (s, 1H; H-6), 7.97e7.94 (m, 2H: fluorene-H), 7.90 (d, J¼7.9 Hz, 1H; fluorene-H), 7.69e7.66 (m, 2H; fluorene-H), 7.49 (d, J¼7.9 Hz, 2H; fluorene-H), 6.14 (t, J¼6.4 Hz, 1H; H-10 ), 5.29 (d, J¼4.3 Hz, 1H; OH30 ), 5.22 (t, J¼4.8 Hz, 1H; OH-50 ), 4.28e4.25 (m, 1H; H-30 ), 3.98 (s, 2H; ArCH2), 3.82 (q, J¼3.2 Hz, 1H; H-40 ), 3.70e3.57 (m, 2H; H-50 ), 2.23e2.12 (m, 2H; H-20 ); 13C NMR (100 MHz, DMSO-d6): d 161.5, 154.5 (d, 1JCF¼37 Hz), 149.5, 144.4, 143.7, 143.5, 140.9, 137.9, 135.6, 130.1, 127.8, 120.9, 120.4, 120.2, 120.1, 117.9, 115.9 (d, 2JCF¼289 Hz), 98.4, 92.6, 87.6, 84.9, 82.6, 70.0, 60.9, 36.5; 19F NMR (471 MHz, DMSO-d6) d 73.8; HRMS-FAB (m/z): [MþNa]þ calcd for C26H20F3N3O6Na, 550.1202; found, 550.1205. 4.2.6. 2 0 -Deoxy-5-(7-amino-9H-fluoren-2-ylethynyl)uridine (UAF). Ammonia solution (3.5 mL) was added to 6 (251 mg, 0.476 mmol) in MeOH (1.8 mL) and then the mixture was heated under reflux for 20 h. After evaporation of the solvent in vacuo, the residue was recrystallized (MeOH) to give a solid (145 mg, 71%): mp>207 C dec; IR (film): n 3294, 3160, 3040, 2897, 2823, 2212, 1694, 1457, 1273, 1193, 1095, 990, 816, 689, 609 cm1; 1H NMR (400 MHz, DMSO-d6): d 11.67 (s, 1H; NH), 8.35 (s, 1H; H-6), 7.61 (d, J¼7.9 Hz, 1H; fluorene-H), 7.54 (d, J¼8.2 Hz, 1H; fluorene-H), 7.51 (s, 1H; fluorene-H), 7.36 (dd, J¼7.8, 1.3 Hz, 1H; fluorene-H), 6.77 (s, 1H; fluorene-H), 6.60 (dd, J¼8.2, 1.5 Hz, 1H; fluorene-H), 6.15 (t, J¼6.6 Hz, 1H; H-10 ), 5.39 (br s, 2H; NH2), 5.27 (d, J¼3.5 Hz, 1H; OH30 ), 5.18 (t, J¼4.6 Hz, 1H; OH-50 ), 4.28e4.24 (m, 1H; H-30 ), 3.82 (q, J¼3.2 Hz, 1H; H-40 ), 3.76 (s, 2H; ArCH2), 3.68e3.58 (m, 2H; H-50 ), 2.22e2.17 (m, 2H; H-20 ); 13C NMR (100 MHz, DMSO-d6): d 161.9, 149.8, 145.6, 143.6, 143.2, 142.3, 130.3, 129.3, 127.7, 121.5, 118.4, 118.0, 113.5, 110.6, 99.1, 93.5, 88.0, 85.1, 81.9, 79.5, 70.4, 61.3, 36.4;
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HRMS-FAB (m/z): [MþH]þ calcd for C24H22N3O5, 432.1561; found, 432.1562. 4.2.7. 20 -Deoxy-5-(7-N,N-dimethylamino-9H-fluoren-2-ylethynyl) uridine (UDAF). (PPh3)2PdCl2 (9.90 mg, 0.0141 mmol) and CuI (2.70 mg, 0.0141 mmol) were added to a solution of 20 -deoxy-5iodouridine (49.8 mg, 0.141 mmol) and 5 (49.2 mg, 0.211 mmol) in Et3N (0.6 mL) and DMF (1.8 mL). After Ar had been bubbled through the solution for 2 min, the mixture was subjected to 10 pump/purge cycle and then it was stirred at 50 C for 4 h. After evaporation of the solvent in vacuo, the residue was subjected to column chromatography (SiO2; CH2Cl2/MeOH, 40:1) to yield a solid (30.0 mg, 46%): mp>244 C dec; IR (film): n 3838, 3739, 3614, 3377, 3161, 3046, 2808, 2360, 2206, 1672, 1609, 1502, 1457, 1356, 1275, 1200, 1096, 992, 924, 800, 701, 652, 601 cm1; 1H NMR (400 MHz, DMSO-d6): d 11.68 (s, 1H; NH), 8.36 (s, 1H; H-6), 7.70 (d, J¼3.8 Hz, 1H; fluorene-H), 7.68 (d, J¼3.1 Hz, 1H; fluorene-H), 7.54 (s, 1H; fluorene-H), 7.39 (d, J¼7.9 Hz, 1H; fluorene-H), 6.95 (s, 1H; fluorene-H), 6.77 (dd, J¼8.5, 2.0 Hz, 1H; fluorene-H), 6.14 (t, J¼6.5 Hz, 1H; H-10 ), 5.27 (d, J¼4.2 Hz, 1H; OH-30 ), 5.19 (t, J¼4.8 Hz, 1H; OH-50 ), 4.28e4.27 (m, 1H; H-30 ), 3.84 (s, 2H; ArCH2), 3.82e3.80 (m, 1H; H-40 ), 3.69e3.57 (m, 2H; H-50 ), 2.97 (s, 6H; NCH3), 2.22e2.12 (m, 2H; H-20 ); 13C NMR (100 MHz, DMSO-d6): d 161.9, 150.8, 149.8, 145.6, 143.7, 142.9, 142.5, 130.4, 129.4, 127.8, 121.4, 118.8, 118.3, 111.8, 109.0, 99.0, 93.5, 88.0, 85.2, 82.0, 79.5, 70.4, 61.2, 36.7; HRMS-FAB (m/z): [MþH]þ calcd for C26H26N3O5, 460.1874; found, 460.1870.
bromide (MTT) assay (Sigma) according to the manufacturer’s instruction. 4.7. Fluorescence imaging HeLa human cervical cancer cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; WelGENE Inc., Korea) containing 10% fetal bovine serum (FBS) and 1% antibiotics and were grown in a humidified incubator at 37 C in an atmosphere containing 5% CO2. For experiments, HeLa cells (2105) were plated in six-well culture plates and incubated with DMEM containing 10% FBS at 37 C under humidified conditions with 5% CO2 for 24 h. A solution of nucleoside (UAF or UDAF, 10 mM) in phenol red-free DMEM containing 2% FBS was added to each well, and the HeLa cells were then incubated at 37 C for 18 h. After incubation, the supernatant was removed, and the cells were washed twice with PBS. Phenol redfree DMEM containing 2% FBS (pH adjusted to 4.2 or 7.3) was added. After incubation for 2 h, we recorded the fluorescence images. Acknowledgements This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014R1A1A2056281 and NRF2012R1A1A2044945) and KOSEF by MEST (NRF-2011-0012830). Supplementary data
4.3. UV absorption measurements Ultraviolet (UV) spectra were recorded at various values of pH in 10 mM phosphate buffer (0.16 M NaCl) at 25 C using a Cary 100 UVevis spectrophotometer and 10-mm-path quartz cell, with respect to a pure-solvent reference. All samples were prepared from a stock solution in DMSO to ensure solubility, and hence, all samples contain 0.5% DMSO. 4.4. Fluorescence experiments All samples were prepared as described above for the UV absorption measurements. Fluorescence spectra were recorded using a Cary Eclipse fluorescence spectrophotometer (cell path length: 1 cm; excitation at 260 nm for FL, 270 nm for AF, 290 nm for DAF, 330 nm for UFL, 330 nm for UAF and 350 nm for UDAF). The excitation and emission bandwidth was 1 nm. The fluorescence quantum yields (FF) were determined using solution of pyrene in cyclohexane or fluorene in EtOH as a reference.21 The pKa value was determined by interpolation of a Boltzmann sigmoidal curve between the normalized fluorescence intensity versus pH. 4.5. pH cycling and selectivity experiments The pH cycling and selectivity experiments were performed at room temperature. UAF or UDAF (2.5 mM) was cycled between pH 3.5 and 7.3 by alternate addition of 5.3 mL 5 M HCl and 5.8 mL of 5 M NaOH, respectively. The stock solutions of metal ions and amino acids were prepared from KCl, NaCl, CaCl2, MgSO4, CdSO4$H2O, CuCl2$2H2O, CoCl2$6H2O, MnCl2$4H2O, NiCl2$6H2O, L-Glutamic acid, L-Cystein, L-Histidine, L-Proline, L-Phenylalanine, L-Glycine, LAlanine, L-Valine, L-Methionine. 4.6. MTT assay for the cell cytotoxicity The cytotoxicity measurements were evaluated in a HeLa cell line by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
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