Acetylated Trifluoromethyl Diboronic Acid Anthracene with a Large Stokes Shift and Long Excitation Wavelength as a Glucose-Selective Probe
Acetylated Trifluoromethyl Diboronic Acid Anthracene with a Large Stokes Shift and Long...
Choi, Hongsik;Song, Inhyeok;Park, Chul Soon;Yim, Heung-seop;Kim, Joong Hyun
2022-03-08 00:00:00
applied sciences Article Acetylated Trifluoromethyl Diboronic Acid Anthracene with a Large Stokes Shift and Long Excitation Wavelength as a Glucose-Selective Probe † † Hongsik Choi , Inhyeok Song , Chul Soon Park, Heung-seop Yim * and Joong Hyun Kim * Drug Manufacturing Center, Daegu-Gyeongbuk Medical Innovation Foundation, 80 Chumbok-ro, Dong-gu, Daegu City 41061, Korea; hschoi@kmedihub.re.kr (H.C.); sih1222@kmedihub.re.kr (I.S.); cspark@kmedihub.re.kr (C.S.P.) * Correspondence: yimhs@kmedihub.re.kr (H.-s.Y.); jhkim@kmedihub.re.kr (J.H.K.) † These authors contributed equally to this work. Featured Application: Glucose detection such as in-vivo continuous glucose monitoring or point- of-care detection. Abstract: Continuous control of blood glucose levels is important for the effective treatment of diabetes. The short-term use of enzymatic continuous monitoring systems involves expensive maintenance and is inconvenient, which limits their widespread use by diabetes patients. The fluorescent diboronic anthracene-embedded system has demonstrated in vivo continuous glucose monitoring for 12 times longer than enzymatic systems by protecting the dye from reactive oxygen species. However, its small Stokes shift and low excitation and emission wavelength should be heavily considered for easy fabrication. We successfully synthesized a derivative of bis-phenyl boronate with a large Stokes shift and long excitation wavelength by adding an acetyl moiety to the anthracene ring. Citation: Choi, H.; Song, I.; Park, C.S.; This resulted in a ~90-nm Stokes shift and 15-nm and 80-nm redshifts of the excitation and emission Yim, H.-s.; Kim, J.H. Acetylated wavelengths, respectively. The fluorescence of the synthesized probe increased proportionally with Trifluoromethyl Diboronic Acid the glucose concentration because the formation of the boronic acid-glucose complex prevented Anthracene with a Large Stokes Shift photoinduced electron transfer. The association constant and quantum yield for acetyl-substituted and Long Excitation Wavelength as a diboronic anthracene with glucose was 20% and 13% higher than that of the analog, respectively. Glucose-Selective Probe. Appl. Sci. While keeping resistance to the oxidation by reactive oxygen species, the improved optical properties 2022, 12, 2782. https://doi.org/ 10.3390/app12062782 and glucose-detecting performances of the newly synthesized dye will allow better pairing of the source and detecting unit for in vivo continuous glucose monitoring, leading to easy fabrication and Academic Editor: Emmanuel then contributing more to utilization by diabetes patients. Stratakis Received: 8 February 2022 Keywords: diboronic acids; stokes shift; photoinduced energy transfer; diabetes; glucose sensor Accepted: 4 March 2022 Published: 8 March 2022 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in published maps and institutional affil- Control of blood glucose levels is extremely important for patients with diabetes [1–4]. iations. However, unpredictable fluctuations in blood glucose levels and variations between in- dividual patients make it difficult to effectively treat diabetes [5,6]. Particularly, special care is required for insulin-dependent diabetes patients because insulin injection can some- times result in hypoglycemia, leading to coma or death. Therefore, continuous glucose Copyright: © 2022 by the authors. monitoring (CGM) is recommended for such patients. Four CGM systems are available on Licensee MDPI, Basel, Switzerland. the market [7]. Most of these systems are enzyme-immobilized needle-injection systems. This article is an open access article The high maintenance cost and inconvenience of replacing the sensors every 6–14 days is distributed under the terms and the main hurdle for their widespread use [8]. Globally, a very low percentage of patients conditions of the Creative Commons with diabetes who require CGM use the systems worldwide [9]. While there have been Attribution (CC BY) license (https:// great efforts to increase the instability of enzymes, the period of use of the enzymatic creativecommons.org/licenses/by/ CGM systems has been not increased for a long time [10,11]. The one non-enzymatic CGM 4.0/). Appl. Sci. 2022, 12, 2782. https://doi.org/10.3390/app12062782 https://www.mdpi.com/journal/applsci Appl. Sci. 2022, 12, 2782 2 of 12 system relies on a diboronic acid-based fluorescent molecule. For the specific and reversible interaction of diboronic acids with glucose, several derivatives have allowed in vivo glu- cose monitoring [12–17]. The immune response to the implanted device generates reactive oxygen species (ROS) such as H O [18]. Therefore, resistance to ROS is highly important 2 2 for the long-term use of CGM. The addition of an electron acceptor group to the aromatic moieties with boronate residues increased the resistance to oxidation by ROS [19]. For example, trifluoromethyl-substituted dibornic acid anthracene resulted in a 24-fold increase in the half-life for 10 M of H O [20]. Additionally, the fluorescent probe under the skin 2 2 can be protected against reactive oxygen species (ROS) by a platinum coating. As the result, the non-enzymatic implantable system can be used for up to 180 days [13]. However, the excitation and emission spectrum of the dye are largely overlapped. The maximum excita- tion and emission wavelengths of the used trifluoromethyl derivative were approximately 405 nm and 430 nm, respectively. The largely overlapped spectra and the small Stokes shift make it difficult to select a light source and detector pair. To minimize the noise resulting from the overlapped spectra, the system needs to use a higher excitation energy source than the dye’s maximum excitation and an additional photodiode as a reference, aside from the main one [21]. The lower excitation wavelength can result in more heat generation and energy consumption. Therefore, improvement of the optical characteristics, such as a large Stokes shift and longer excitation wavelength, is still in demand. In this study, we report the synthesis of a new diboronic anthracene with a larger Stokes shift and longer excitation wavelength than the commercially used analog. It is known that the acetyl group can increase the emission wavelength of the dye [22,23]. By introducing the acetyl group on the anthracene, we could increase the Stokes shift up to 90 nm. The introduced acetyl group did not affect the resistance to ROS. Instead, the acetylation resulted in an improved glucose detecting ability for the dye. 2. Materials and Methods 2.1. Materials The 9,10-dimethylanthracene (98%) was purchased from Angel Pharmatech Ltd. (Shanghai, China). The -alanine t-butyl ester hydrochloride, n-(3-aminopropyl) methacry- lamide hydrochloride (97%) was purchased from Leap Labchem Co. (Hong Kong, China). Other starting materials for the synthesis and solvents were purchased from Aldrich, TCI, and Alfa Aesar and used without further purification. Fluorescein, D-(+) galactose (98%), D-mannitol (98%), D-(+) maltose (99%), sucrose (99.5%), and ibuprofen (98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). D-glucose (98%), D-fructose (99%), and lactose were purchased from Junsei (Saitama, Japan). Dimethyl sulfoxide (99%) was purchased from Duksan (Gyeonggi-do, Korea). Sodium hydroxide (97%) was purchased from Daejung (Gyeonggi-do, Korea). PBS (10X) was purchased from GenDEPOT (Katy, TX, USA). 2.2. Methods 2.2.1. Measurement of Saccharide-Dependent Fluorescence Stock solutions were prepared by dissolving 40-mM dyes in DMSO and storing them at 4 C before use. The stock solution was diluted in 1 PBS (pH 7.4) to obtain 20 M of the dye in the buffer. The dye solution was mixed with an equal volume of the saccharide solution. The saccharide mixture (200 L) was transferred to a black 96-well plate (SPL Life Science Co., Ltd., Pocheon-si, Korea), and the fluorescence was measured using a SpectraMaxiD3 (Molecular Devices, San Jose, CA, USA). 2.2.2. Measurement of Decay Time The dye (20 M) in the buffer containing 200 mg/dL glucose was prepared from the stock solution. The time-resolved fluorescence intensity at 490 nm of the mixture (200 L) in the 96-well plate was measured every 30 s after adding 10 mM H O to the mixture 2 2 using the well plate reader. Appl. Sci. 2022, 12, x FOR PEER REVIEW 3 of 13 The dye (20 μM) in the buffer containing 200 mg/dL glucose was prepared from the stock solution. The time-resolved fluorescence intensity at 490 nm of the mixture (200 μL) in the 96-well plate was measured every 30 s after adding 10 mM H2O2 to the mixture using the well plate reader. Appl. Sci. 2022, 12, 2782 3 of 12 2.2.3. Relative Quantum Yield The emission spectra of AcTDA and TDA in SpectraMaxiD3 1× PBS were recorded. 2.2.3. Relative Quantum Yield For the relative quantum yield, the emission spectra of fluorescein at the various The emission spectra of AcTDA and TDA in SpectraMaxiD3 1 PBS were recorded. concentrations in 0.1 M NaOH were also measured at 360 nm and 340 nm of excitation for For the relative quantum yield, the emission spectra of fluorescein at the various concentra- AcTDA and TDA, respectively. The area of the emission spectra of each dye at the tions in 0.1 M NaOH were also measured at 360 nm and 340 nm of excitation for AcTDA measured concentrations (1 μM, 5μM, 10 μM, and 20 μM) were plotted with the and TDA, respectively. The area of the emission spectra of each dye at the measured concen- corresponding absorbance at 360 nm and 340 nm for AcTDA and TDA, respectively. trations (1 M, 5M, 10 M, and 20 M) were plotted with the corresponding absorbance at 360 nm and 340 nm for AcTDA and TDA, respectively. 3. Results 3. Results 3.1. Synthesis 3.1. Synthesis The acetylated diboronic anthracenes were prepared according to the synthetic The acetylated diboronic anthracenes were prepared according to the synthetic routes routes shown in Scheme 1. The detailed synthetic procedure is described below. shown in Scheme 1. The detailed synthetic procedure is described below. O O B B O O O O Br B B O O F C 3 NBS Br CH3COOK F C AIBN F C 3 3 Pd(dppf)Cl2 1,2-dichloroethane Dioxane Br Acetyl chloride NBS AlCl3 1,2-dichloroethane MC Br O OH CF O O O O CF O NH 2 B O N Br HN N F C N NH HO B 2 HO 2 OH OH HO OH R B OH DIPEA DIPEA OH TFA N BHT O O BHT MC NH N CHCl3 CHCl3 CF HN CF HN HN O O AcTDA(7a) : R = COCH TDA(7b) : R = H Scheme 1. Synthesis route of acetylated trifluoromethyl diboronic anthracene (AcTDA). Scheme 1. Synthesis route of acetylated trifluoromethyl diboronic anthracene (AcTDA). 3.1.1. 4,4,5,5-Tetramethyl-2-(2-methyl-4-(trifluoromethyl)phenyl)-1,3,2-dioxoborolane 3.1.1. 4,4,5,5-tetramethyl-2-(2-methyl-4-(trifluoromethyl)phenyl)-1,3,2-dioxoborolane (Compound 1) (Compound 1) Compounds of 1-bromo-2-methyl-4-(trifluoromethyl) benzene (20.00 g, 0.084 mol), 0 0 0 0 0 4,4,4 ,4 ,5,5,5 ,5 -octamethyl-2,2 -bis(1,3,2-dioxoborolane) (42.5 g, 0.167 mol), potassium ac- Compounds of 1-bromo-2-methyl-4-(trifluoromethyl) benzene (20.00 g, 0.084 mol), etate (32.85 g, 0.335 mol), and [1,1’bis(diphenylphosphino)ferrocene]dichloropalladium(II) 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bis(1,3,2-dioxoborolane) (42.5 g, 0.167 mol), potassium (6.12 g, 0.0084 mol) in 600 mL of dioxane were stirred at 100 C for 1 h. After the solution acetate (32.85 g, 0.335 mol), and was cooled to room temperature, 600 mL of DI water was added into the mixture, and the [1,1’bis(diphenylphosphino)ferrocene]dichloropalladium(II) (6.12 g, 0.0084 mol) in 600 mixture was extracted using methylene chloride (600 mL). The organic layer of the mixture mL of dioxane were stirred at 100 °C for 1 h. After the solution was cooled to room was separated and dried over anhydrous magnesium sulfate. The dried compound was temperature, 600 m stirred L of in 500 DI wat mL ofe hexane r was ad for 0.5 dehd until into the mixture, and the m the formation of precipitation.iOily xture was compound 1 (21.5 g, 89.9%) was obtained after the formed precipitate was filtered off and purified using extracted using methylene chloride (600 mL). The organic layer of the mixture was silica gel chromatography with dichloromethane/hexane (1:8, v/v) as the eluent ( H NMR separated and dried over anhydrous magnesium sulfate. The dried compound was stirred (400 MHz, chloroform-d): 7.88(d, 1H), 7.43–7.41(m, 2H), 2.61(s, 3H), and 1.38(s, 12H)). in 500 mL of hexane for 0.5 h until the formation of precipitation. Oily compound 1 (21.5 Appl. Sci. 2022, 12, 2782 4 of 12 3.1.2. 2-(2-(Bromomethyl)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxoborolane (Compound 2) 4,4,5,5-tetramethyl-2-(2-methyl-4-(trifluoromethyl)phenyl)-1,3,2-dioxoborolane (21.5 g, 0.075 mol) and N-bromosuccinimide (NBS) (14.7 g, 0.083 mol) were dissolved in 390 mL of 1,2-dichloroethane, and 2,2-azobisisobutyronitrile (AIBN) (1.23 g, 0.075 mol) was added into the mixture and stirred at 80 C for 3 h before the mixture was allowed to cool to RT, followed by filtration. After removal of the solvent under reduced pressure, the mixture was stirred in 200 mL of hexane for 0.5 h. Yellow oily compound 2 was obtained after evaporating the solvent of the filtered-off precipitate under a vacuum (yield (29.00 g, 10.6%); H NMR (400 MHz, chloroform-d): 7.93(d, 1H), 7.63(s, 1H), 7.52(d, 1H), 4.92(s, 2H), and 1.39(s, 12H)). 3.1.3. 2-Acetyl-9,10-dimethylanthracene (Compound 3) 9,10-dimethylanthracene (20.0 g, 0.1 mol) was dissolved in methylene chloride (600 mL). Acetyl chloride (0.4 g, 0.12 mol) and 18.8 g (0.149 mol) of aluminum chloride were added into the mixture and stirred at 0~5 C. After 5 h of additional stirring at RT and refluxing for 1 h, the mixture was allowed to cool to RT. Then, 1 kg of ice and 50 mL of hydrochloric acid were added into the reaction mixture. The mixture was extracted with methylene chloride and water. After removal of the solvent under reduced pressure, the product was purified using silica gel chromatography with ethyl acetate/hexane (1:10, v/v) as the eluent. Compound 3 was obtained as a yellow powder in 56.5% yield (14.0 g); H NMR (400 MHz, chloroform-d): 9.00(m, 7H), 3.15(d, 6H), and 2.80(s, 3H). 3.1.4. 2-Acetyl-9,10-bis(bromomethyl)anthracene (Compound 4) 2-acetyl-9,10-dimethylanthracene (14.0 g, 0.056 mol) was dissolved in 1,2-dichloroethane (300 mL). After the complete addition of N-bromosuccinimide (22.1 g, 0.124 mol), the reaction solution was stirred for 1 h at 85 °C. The reaction mixture was allowed to cool to RT, and the solvent was evaporated under reduced pressure. After the mixture was stirred in 150 mL of methanol for 0.5 h, the precipitate was filtered, washed with methanol, and dried under a vacuum at 40 C for 12 h. The obtained compound (compound 4) was a bright yellow powder (yield: 7.7 g, 33.6%; H NMR (400 MHz, chloroform-d): 9.00–7.50(m, 7H), 5.50(d, 4H), and 2.80(s, 3H)). 3.1.5. Tert-butyl 3-(([3-acetyl-10-(([3-methacrylamidopropyl]amino)methyl)anthracen-9- yl]methyl)amino)propanoate (Compound 5) Compound 4 (7.7 g, 0.019 mol) consisted of -alanine t-butyl ester hydrochloride (12.5 g, 0.069 mol), n-(3-aminopropyl) methacrylamide hydrochloride (12.3 g, 0.069 mol), butylated hydroxytoluene (BHT) (0.28 g, 0.00127 mol), and NN-diisopropylethylamine (DIPEA) (24 mL, 0.138 mol) in 400 mL of chloroform dissolved at 30 C for 18 h. After the solvent was evaporated under reduced pressure, the mixture was washed with distilled water (3 times 200 mL) and dried over MgSO . The obtained crude product was purified using silica chromatography with CHCl /MeOH (95:5, v/v) with the eluent. A yellow powder was obtained (yield: 1.1 g, 15.0%; H NMR (400 MHz, chloroform-d): 8.70–7.29(m, 8H), 5.79(s, 1H), 5.72(s, 1H), 4.16(m, 6H), 3.18(d, 2H), 2.94(d, 2H), 2.50(m, 7H), 1.98(s, 3H), 1.74(t, 2H), and 1.42(s, 9H); LC-MS: calculated for C H N O m/z, 531.70; found m/z: 32 41 3 4 532 [M + H]+). 3.1.6. (2-([((3-Acetyl-10-[((2-borono-5-[trifluoromethyl]benzyl)[3-methacrylamidopropyl] amino)methyl)anthracen-9-yl]methyl)(3-[tert-butoxy]-3-oxopropyl)amino)methyl]-4- (trifluoromethyl)phenyl)boronic Acid (Compound 6) Compound 5 (0.2 g, 0.38 mmol) and NN-diisopropylethylamine (DIPEA) (520 mL, 2.97 mmol) in 20 mL of chloroform were stirred at RT. Butylated hydroxytoluene (BHT) (0.01 g, 0.038 mmol), 2-(2-(bromomethyl)-4-(trifluoromethyl) phenyl)-4,4,5,5-tetramethyl-1, and 3,2-dioxoborolane (0.535 g, 1.47 mmol) were added into the reaction mixture, followed by stirring at RT for at least 24 h. After removal of the solvent-reduced pressure, the mixture Appl. Sci. 2022, 12, 2782 5 of 12 was dissolved in 20 mL of isopropyl ether and washed 3 times with 20 mL of phosphate buffer (0.2 M, pH 7.0). After removal of the solvent, the product was purified using silica gel chromatography, with CHCl /MeOH (98:2, v/v) as the eluent. A yellow powder was obtained in a 48.3% yield (0.2 g) ( H NMR (400 MHz, chloroform-d): 8.70–7.28(m, 14H), 5.79(s, 1H), 4.20(s, 4H), 4.10(s, 2H), 3.76–3.66(m, 6H), 3.18(d, 2H), 2.46(m, 7H), 1.98(s, 3H), 1.73(t, 2H), and 1.42(s, 9H); LC-MS: calculated for C H B F N O m/z: 935.58; found 48 53 2 6 3 8 + + m/z: 936 [M + H]+; 918 [M-H O + H] and 900 [M-2H O + H] ). 2 2 3.1.7. 3-(((3-Acetyl-10-(((2-borono-5-(trifluoromethyl)benzyl)(3-methacrylamidopropyl) amino)methyl)anthracen-9-yl)methyl)(2-borono-5-(trifluoromethyl)benzyl)amino) propanoic Acid (Compound 7a) Compound 6 (0.2 g, 0.182 mmol) in 5 mL of methylene chloride containing 20 wt% trifluoroacetic acid was stirred at room temperature for 20 h. After evaporating the sol- vent under a vacuum, the product was purified using silica gel chromatography with CHCl /MeOH (95:5, v/v) as the eluent. A yellow powder was obtained in a 43.5% yield (0.074 g) ( H NMR (400 MHz, chloroform-d): 12.27(s, 1H), 8.70–7.28 (m, 14H), 5.79(s, 1H), 4.20(s, 4H), 4.10(s, 2H), 3.76–3.66(m, 6H), 3.18(d, 2H), 2.46(m, 7H), 1.98(s, 3H), 1.73(t, 2H), and 1.42(s, 9H); LC-MS: calculated for C H B F N O m/z: 879.47; found m/z: 880 [M + 44 45 2 6 3 8 + + H]+; 862 [M-H O + H] , 844[M-2H O + H] ). 2 2 3.1.8. 3-((2-Borono-5-(trifluoromethyl)benzyl)((10-(((2-borono-5-(trifluoromethyl)benzyl) (3-methacrylamidopropyl)amino)methyl)anthracen-9-yl)methyl)amino)propanoic acid (Compound 7b) After carrying out the same procedure, compound 7b was synthesized through the same steps except for acetylation. A yellow powder was obtained in a 51.2% yield (0.089g) ( H NMR (400 MHz, chloroform-d): 8.28–7.38(m, 14H), 5.35(s, 1H), 5.05(s, 1H), 4.95(t, 1H), 4.55(s, 2H), 4.44(s, 2H), 4.35(s, 2H), 4.05(s, 2H), 2.92(m, 2H), 2.87(m, 2H), 2.44(t, 2H), 2.31(t, 2H), 1,58–1.60(m, 5H), 1.38(s, 12H), and 1.34(s, 12H); LC-MS: calculated for C H B F N O m/z: 837.43; found m/z: 838 [M + H]+; 820 [M-H O + H] , 802[M-2H O 42 43 2 6 3 7 2 2 + H] ). 3.2. Fluorogenic Optical Properties After synthesizing acetylated trifluoromethyl diboronic anthracenes (AcTDAs) and tri- fluoromethyl diboronic anthracenes (TDAs), we characterized the optical properties of the analogs by measuring and comparing the emission and excitation spectra. Figure 1 shows the spectra for both molecules. AcTDA, which has trifluoromethyl and acetyl groups, ex- hibited excitation and emission wavelengths at 420 nm and 510 nm, respectively (Figure 1a). In contrast, TDA, the counter-diboronic anthracene with no acetyl group, demonstrated the longest excitation and emission wavelengths at 405 nm and 430 nm, respectively. These results clearly demonstrated that the addition of an acetyl group resulted in ~80 nm and 15 nm red-shifted maximum emission and excitation wavelengths, respectively. Table 1 shows the excitation and emission wavelengths of the reported diboronic anthracene deriva- tives. As shown in Table 1, diboronic anthracene with an acetyl group on the anthracene produced a relatively large Stokes shift with a long excitation wavelength. This analysis confirms the increase in the Stokes shift and excitation wavelength by the acetyl group on the anthracene. Among the diboronic anthracene derivatives reported in Table 1, AcTDA exhibited the largest Stokes shift with the longest excitation wavelength [14,23–26]. Com- pared with similar acetylated derivatives [23], AcTDA produced excitation and emission fluorescence that was red-shifted by at least 15 and 22 nm, respectively. It has been reported that the substitution of a trifluoromethyl group on the quinoline ring of thiazole orange led to a redshift of thiazole orange [27]. Therefore, the two electron-withdrawing groups resulted in the largest Stokes shift with the longest excitation and emission wavelengths. Appl. Sci. 2022, 12, x FOR PEER REVIEW 6 of 13 Appl. Sci. 2022, 12, x FOR PEER REVIEW 6 of 13 Appl. Sci. 2022, 12, x FOR PEER REVIEW 6 of 13 excitation wavelength by the acetyl group on the anthracene. Among the diboronic Appl. Sci. 2022, 12, x FOR PEER REVIEW 6 of 13 anthracene derivatives reported in Table 1, AcTDA exhibited the largest Stokes shift with excitation wavelength by the acetyl group on the anthracene. Among the diboronic the longest excitation wavelength [14,23–26]. Compared with similar acetylated Appl. Sci. 2022, 12, x FOR PEER an R thra EVIEW cene deri vatives reported in Table 1, AcTDA exhibited the largest Stokes shift with 6 of 13 excitation wavelength by the acetyl group on the anthracene. Among the diboronic derivatives [23], AcTDA produced excitation and emission fluorescence that was red- the longest excitation wavelength [14,23–26]. Compared with similar acetylated anthracene derivatives reported in Table 1, AcTDA exhibited the largest Stokes shift with excitation wavelength by the acetyl group on the anthracene. Among the diboronic shifted by at least 15 and 22 nm, respectively. It has been reported that the substitution of derivatives [23], AcTDA produced excitation and emission fluorescence that was red- the longest excitation wavelength [14,23–26]. Compared with similar acetylated a a t nthra rifluorome cene deri thyl g vativreoup s rep oonrted i the n qu Ta inol ble 1 ine r , AcTDA exhi ing of thiazole orange le bited the largest Stokes d to a re shi dsfhift t wi of th shifted by at least 15 and 22 nm, respectively. It has been reported that the substitution of derivatiexci ves [2 tation wavel 3], AcTDA ength by produced exci the acta etyl tion group on the and emission fl anthra uorecene. scence Among the that was red- diboronic the l thiazoole o ngest range [27]. Ther excitation wa efore, the two el velength [14,23–2 ectron-w 6]. Compa ithdrawing grou red with si ps re milasu r acetyl lted in t ated he a trifluoromethyl group on the quinoline ring of thiazole orange led to a redshift of shifteda b ny thra at lcene deri east 15 an va d ti 2v 2e nm s rep , respect orted iin vely Table 1 . It has b , AcTDA exhi een reported bited the la that the subst rgest Stokes itution o shi f ft with derivatives [23], AcTDA produced excitation and emission fluorescence that was red- largest Stokes shift with the longest excitation and emission wavelengths. thiazole orange [27]. Therefore, the two electron-withdrawing groups resulted in the a trifluorome the longest thyl grexci oupta otinon wa the quvel inol ength [ ine ring14 ,of thiazole orange le 23–26]. Compared wi d to th si a re mil dsahift r acetyl of ated shifted by at least 15 and 22 nm, respectively. It has been reported that the substitution of largest Stokes shift with the longest excitation and emission wavelengths. thiazole o derirvan atige [27]. Ther ves [23], AcTDA efore, the two el produced exci ectron-w tation and ithdrawing grou emission fluor ps re escence sulted in t that was he red- a trifluoromethyl group on the quinoline ring of thiazole orange led to a redshift of (a) largest shift Stokes shif ed by at t wi least th the l 15 an od ngest 22 nm excit , respect ation and emission ively. It has b een reported wavelengths.that the subst itution of thiazole orange [27]. Therefore, the two electron-withdrawing groups resulted in the Em. Ex. 1.a t0 rifluoromethyl group on the quinoline ring of thiazole orange led to a redshift of (a) largest Stokes shift with the longest excitation and emission wavelengths. Em. Ex. thiazole orange [27]. Therefore, the two electron-withdrawing groups resulted in the Appl. Sci. 2022, 12, 2782 6 of 12 1.0 (a) largest Stokes shift with the longest excitation and emission wavelengths. Em. Ex. 1.0 (a) 0.5 Em. Ex. 1.0 0.5 (a) Em. Ex. 1.0 0.5 0.0 0.5 0.0 300 400 500 600 Wavelength (nm) 300.05 400 500 600 0.0 Wavelength (nm) 300 400 500 600 0.0 Figure 1. Emission (Em.) and excitation (Ex.) spectra of trifluoromethyl diboronic anthracenes (a) Wavelength (nm) 300 400 500 600 with and (b) without an acetyl group. Norm. I. denotes normalized emission and excitation intensity Figure 1. Em0. ission ( 0 Em.) and excitation (Ex.) spectra of trifluoromethyl diboronic anthracenes (a) at the maximum wavelength. Wavelength (nm) with and (b) without an acetyl group. Norm. I. denotes normalized emission and excitation intensity 300 400 500 600 Figure 1. Emission (Em.) and excitation (Ex.) spectra of trifluoromethyl diboronic anthracenes (a) at the maximum wavelength. with and (b) without an acetyl group. Norm. I. denotes normalized emission and excitation intensity Wavelength (nm) Table 1. Emission and excitation wavelength of the reported derivate of diboronic acid Figure 1. Emission (Em.) and excitation (Ex.) spectra of trifluoromethyl diboronic anthracenes (a) at the maximum wavelength. anthracene. with and ( Table 1. Em b)i without an acetyl ssion and excitation wave group. Norm. length I. of the reported de denotes normalized rivate of diboronic acid emission and excitation intensity Figure 1. Emission (Em.) and excitation (Ex.) spectra of trifluoromethyl diboronic anthracenes Figure 1. Emission (Em.) and excitation (Ex.) spectra of trifluoromethyl diboronic anthracenes (a) at the maxi anthracene. mum wavelength. (a) with and (b) without an acetyl group. Norm. I. denotes normalized emission and excitation Structure λex (nm) λem (nm) Ref. Table 1. Emission and excitation wavelength of the reported derivate of diboronic acid with and (b) without an acetyl group. Norm. I. denotes normalized emission and excitation intensity intensity at the maximum wavelength. anthracene. at the maxi Struc mum wavel ture ength. λex (nm) λem (nm) Ref. Table 1. Emission and excitation wavelength of the reported derivate of diboronic acid anthracene. Structure λex (nm) λem (nm) Ref. Table 1. Emission and excitation wavelength of the reported derivate of diboronic acid anthracene. Table 1. Emission and excitation wavelength of the reported derivate of diboronic acid Structure λex (nm) λem (nm) Ref. anthracene. Structure l (nm)400 4 l (nm)80 [24]Ref. ex em Structure 400 4 λex (nm) 80 λem (nm) Ref.[24] 400 480 [24] 400400 4 48080 [24][ 24] 400 480 [24] 375 405/427 [25] 375 405/427 [25] 375375 4 405/42705/427 [25][ 25] 375 405/427 [25] 375 405/427 [25] 370 423 [14] 370370 4 42323 [14][ 14] 370 423 [14] Appl. Sci. 2022, 12, x FOR PEER REVIEW 7 of 13 370 423 [14] 377377 4 42727 [26][ 26] 370 423 [14] 377 427 [26] 377 427 [26] 377 427 [26] 405405 4 48888 [23][ 23] 377 427 [26] 3.3. Measurement of Oxidation Resistance 3.3. Measurement of Oxidation Resistance For the long-term operation of the implanted CGM system, resistance to ROS is For the long-term operation of the implanted CGM system, resistance to ROS is extremely important. As mentioned previously, trifluoromethyl substitution increased extremely important. As mentioned previously, trifluoromethyl substitution increased the resistance to oxidation of the hydroxyl groups of boronate, which interacted with the the resistance to oxidation of the hydroxyl groups of boronate, which interacted with the diol of glucose to form a boronic acid-glucose complex. To determine how the added diol of glucose to form a boronic acid-glucose complex. To determine how the added acetyl group affected the resistance, we measured the changes in the fluorescence intensity acetyl of the two analogs in the presence of hydrogen peroxide, the main ROS generated by group affected the resistance, we measured the changes in the fluorescence intensity of the immune response to the implanted device [20,28–30]. As shown in Figure 2, the two the two analogs in the presence of hydrogen peroxide, the main ROS generated by the immune response to the implanted device [20,28–30]. As shown in Figure 2, the two analogs exhibited identical decay rates of fluorescence intensity. This result indicates that the substituted acetyl group did not affect the oxidation resistance. Figure 2. Comparison of oxidation resistance of AcTDA and TDA. Normalized fluorescence intensity after 4 mM H2O2 was added to 20 μM of (a) AcTDA and (b) TDA with 200 mg/dL glucose in 1× PBS (pH 7.4). 3.4. Glucose-Dependent Optical Characterisitcs Next, we explored the fluorescence change of AcTDA with changes in the glucose concentration by collecting the emission spectra of the compound with various concentrations of glucose. Figure 3 shows the measured emission spectra of AcTDA mixed with glucose. In the presence of glucose, the emission spectra were blue-shifted by ~20 nm, and the intensity increased. According to the mechanism of B-N bond formation, the formation of a boronic acid complex with the diols from a saccharide, such as glucose, created intramolecular interactions between the boronic acids and amines. As shown in Figure 4, the B-N bond prevented the nitrogen lone pair from participating in photoinduced electron transfer (PET) [14,31]. As a result, the quenched anthracene fluorescence was recovered. Thus, we could observe the increased fluorescence intensity of AcTDA as the glucose level of the mixture increased. Next, we measured the association constant of AcTDA with glucose using the modified Benesi–Hildebrand plot [25]: NorNo m.r No F m . I.r No . F m . I.r. F m . .I .F. I. Norm. F. I. Appl. Sci. 2022, 12, x FOR PEER REVIEW 7 of 13 405 488 [23] 3.3. Measurement of Oxidation Resistance For the long-term operation of the implanted CGM system, resistance to ROS is extremely important. As mentioned previously, trifluoromethyl substitution increased the resistance to oxidation of the hydroxyl groups of boronate, which interacted with the diol of glucose to form a boronic acid-glucose complex. To determine how the added acetyl group affected the resistance, we measured the changes in the fluorescence intensity of the two analogs in the presence of hydrogen peroxide, the main ROS generated by the immune response to the implanted device [20,28–30]. As shown in Figure 2, the two Appl. Sci. 2022, 12, 2782 7 of 12 analogs exhibited identical decay rates of fluorescence intensity. This result indicates that the substituted acetyl group did not affect the oxidation resistance. analogs exhibited identical decay rates of fluorescence intensity. This result indicates that the substituted acetyl group did not affect the oxidation resistance. Figure 2. Comparison of oxidation resistance of AcTDA and TDA. Normalized fluorescence intensity after 4 mM H O was added to 20 M of (a) AcTDA and (b) TDA with 200 mg/dL glucose in 1 PBS Figure 2. Comparison of oxidation resistance of AcTDA and TDA. Normalized fluorescence 2 2 (pH 7.4). intensity after 4 mM H2O2 was added to 20 μM of (a) AcTDA and (b) TDA with 200 mg/dL glucose in 1× PBS (pH 7.4). 3.4. Glucose-Dependent Optical Characterisitcs Next, we explored the fluorescence change of AcTDA with changes in the glucose con- 3.4. Glucose-Dependent Optical Characterisitcs centration by collecting the emission spectra of the compound with various concentrations of glucose. Figure 3 shows the measured emission spectra of AcTDA mixed with glucose. Next, we explored the fluorescence change of AcTDA with changes in the glucose In the presence of glucose, the emission spectra were blue-shifted by ~20 nm, and the concentration by collecting the emission spectra of the compound with various intensity increased. According to the mechanism of B-N bond formation, the formation of a boronic acid complex with the diols from a saccharide, such as glucose, created intramolec- concentrations of glucose. Figure 3 shows the measured emission spectra of AcTDA ular interactions between the boronic acids and amines. As shown in Figure 4, the B-N mixed with glucose. In the presence of glucose, the emission spectra were blue-shifted by bond prevented the nitrogen lone pair from participating in photoinduced electron transfer ~20 nm, and the intensity increased. According to the mechanism of B-N bond formation, (PET) [14,31]. As a result, the quenched anthracene fluorescence was recovered. Thus, we could observe the increased fluorescence intensity of AcTDA as the glucose level of the the formation of a boronic acid complex with the diols from a saccharide, such as glucose, mixture increased. Next, we measured the association constant of AcTDA with glucose created intramolecular interactions between the boronic acids and amines. As shown in using the modified Benesi–Hildebrand plot [25]: Figure 4, the B-N bond prevented the nitrogen lone pair from participating in 1 1 1 photoinduced electron transfer (PET) [14,31]. As a result, the quenched anthracene = + (1) F F Ka (F F ) [Glucose] F F max max 0 0 0 fluorescence was recovered. Thus, we could observe the increased fluorescence intensity where F is the measured fluorescence intensity, F is the fluorescence intensity of free of AcTDA as the glucose level of the mixture increased. Next, we measured the association AcTDA, and F is the saturated fluorescence intensity of the AcTDA–glucose complex. max constant of AcTDA with glucose using the modified Benesi–Hildebrand plot [25]: We plotted 1/[F F ] versus 1/[glucose] and calculated the association constant for the obtained linear relationship (Figure 3d). The obtained association constant with glucose was 730 18 M . We also measured the association constant of TDA with glucose. Figure 5 shows the fluorescence changes for TDA, depending on the quantity of added glucose. As shown in Figure 5, the fluorescence intensity of TDA increased proportionally with an increasing glucose concentration. The calculated association constant was 606 63 M , which was approximately 20% lower than that of AcTDA. As shown in Figure 5c, no significant spectral shift was observed for glucose mixed with TDA. Instead, the emission peak became narrower. Because the portion of fluorescence from the unbound AcTDA that contributed to the association calculation was smaller than that of TDA, AcTDA could have a higher binding affinity for glucose than TDA. Meanwhile, there was no shift in the excitation spectrum of either dye by glucose (Figure S1). The excitation intensity near 300 nm was decreased for both dyes in the presence of glucose because the contribution of the plastic well plate to the excitation decreased. In addition, we compared the quantum Appl. Sci. 2022, 12, 2782 8 of 12 yield of the two analogs by measuring the relative quantum yield [32]. The relative quantum yield is calculated using a flowing equation: m n r s Q = Q (2) S R m n s r where Q is the quantum yield, m is a gradient of the plot of the integrated fluorescence against absorbance, n is the refractive index of the solvent, and s and r are the sample and reference, respectively. In the equation, the quantum yield of reference is independent of the excited wavelength, but the ratio of the plot of integrated fluorescence against the absorbance is a function of the exciting wavelength [30]. Then, Equation (2) can be rearranged to the ratio of the quantum yield between two samples as follows: Appl. Sci. 2022, 12, x FOR PEER REVIEW 8 of 13 Q m m S1 r1 r2 = (3) Q m m S2 s1 s2 Figure 3. Glucose binding properties of AcTDA. (a) Emission spectra and (b) normalized emission Figure 3. Glucose binding properties of AcTDA. (a) Emission spectra and (b) normalized emission spectra for glucose at concentrations of 0–500 mg/dL. Fluorescence intensity at the maximum wave- spectra for glucose at concentrations of 0–500 mg/dL. Fluorescence intensity at the maximum length was used for the normalization. (c) Relative fluorescence intensity. (d) Benesi–Hildebrand plot. wavelength was used for the normalization. (c) Relative fluorescence intensity. (d) Benesi– Hildebrand plot. Figure 4. Glucose-dependent fluorescence change for AcTDA. Quenched fluorescence of AcTDA was recovered after formation of AcTDA–glucose because photoinduced electron transfer was blocked by the B-N bond pair. 1 1 1 (1) 𝐹 𝐹 𝐾𝑎 𝐹 𝐹 𝑒𝑐𝑢𝑜𝑠𝑙𝐺 𝐹 𝐹 where F is the measured fluorescence intensity, F0 is the fluorescence intensity of free AcTDA, and Fmax is the saturated fluorescence intensity of the AcTDA–glucose complex. We plotted 1/[F–F0] versus 1/[glucose] and calculated the association constant for the obtained linear relationship (Figure 3d). The obtained association constant with glucose −1 was 730 ± 18 M . We also measured the association constant of TDA with glucose. Figure 5 shows the fluorescence changes for TDA, depending on the quantity of added glucose. As shown in Figure 5, the fluorescence intensity of TDA increased proportionally with an Appl. Sci. 2022, 12, x FOR PEER REVIEW 8 of 13 Figure 3. Glucose binding properties of AcTDA. (a) Emission spectra and (b) normalized emission spectra for glucose at concentrations of 0–500 mg/dL. Fluorescence intensity at the maximum Appl. Sci. 2022, 12, 2782 9 of 12 wavelength was used for the normalization. (c) Relative fluorescence intensity. (d) Benesi– Hildebrand plot. Appl. Sci. 2022, 12, x FOR PEER REVIEW 9 of 13 −1 increasing glucose concentration. The calculated association constant was 606 ± 63 M , which was approximately 20% lower than that of AcTDA. As shown in Figure 5c, no significant spectral shift was observed for glucose mixed with TDA. Instead, the emission peak became narrower. Because the portion of fluorescence from the unbound AcTDA that contributed to the association calculation was smaller than that of TDA, AcTDA could have a higher binding affinity for glucose than TDA. Meanwhile, there was no shift in the excitation spectrum of either dye by glucose (Figure S1). The excitation intensity near 300 nm was decreased for both dyes in the presence of glucose because the contribution of the plastic well plate to the excitation decreased. In addition, we compared the quantum yield Figure 4. Glucose-dependent fluorescence change for AcTDA. Quenched fluorescence of AcTDA was Figure 4. Glucose-dependent fluorescence change for AcTDA. Quenched fluorescence of AcTDA of the two analogs by measuring the relative quantum yield [32]. The relative quantum recovered after formation of AcTDA–glucose because photoinduced electron transfer was blocked by was recovered after formation of AcTDA–glucose because photoinduced electron transfer was yield is calculated using a flowing equation: the B-N bond pair. blocked by the B-N bond pair. 1 1 1 (1) 𝐹 𝐹 𝐾𝑎 𝐹 𝐹 𝑒𝑐𝑢𝑜𝑠𝑙𝐺 𝐹 𝐹 where F is the measured fluorescence intensity, F0 is the fluorescence intensity of free AcTDA, and Fmax is the saturated fluorescence intensity of the AcTDA–glucose complex. We plotted 1/[F–F0] versus 1/[glucose] and calculated the association constant for the obtained linear relationship (Figure 3d). The obtained association constant with glucose −1 was 730 ± 18 M . We also measured the association constant of TDA with glucose. Figure 5 shows the fluorescence changes for TDA, depending on the quantity of added glucose. As shown in Figure 5, the fluorescence intensity of TDA increased proportionally with an Figure 5. Figure 5. Glucose-binding Glucose-binding properties properties ofof TDA. TD(A a). Emission (a) Emissi spectra. on spec (b tra. ) Normalized (b) Normalize emission d emission spectra. spectra. Fluorescence intensity at the maximum wavelength was used for the normalization. (c) Fluorescence intensity at the maximum wavelength was used for the normalization. (c) Relative Relative fluorescence intensity. (d) Benesi–Hildebrand plot. fluorescence intensity. (d) Benesi–Hildebrand plot. 3.5. Interference-Dependent Fluorescence 𝑚 𝑛 𝑄 𝑄 (2) To use AcTDA as a glucose probe, it needs to demonstrate high specificity for glucose 𝑚 𝑛 over other interferences, such as six-carbon sugars and common drugs. Therefore, we where Q is the quantum yield, m is a gradient of the plot of the integrated fluorescence measured the fluorescence changes of AcTDA with six different saccharides, including against absorbance, n is the refractive index of the solvent, and s and r are the sample and fructose, galactose, lactose, maltose, mannitol, and sucrose, and the most common drugs. reference, respectively. In the equation, the quantum yield of reference is independent of the excited wavelength, but the ratio of the plot of integrated fluorescence against the absorbance is a function of the exciting wavelength [30]. Then, Equation (2) can be rearranged to the ratio of the quantum yield between two samples as follows: 𝑄 𝑚 𝑚 (3) 𝑄 𝑚 𝑚 Appl. Sci. 2022, 12, x FOR PEER REVIEW 10 of 13 Using fluorescein as the reference, we obtained the gradient of each compound in the presence of 30 mM of glucose (Figure 6). According to Equation (3), the glucose- Appl. Sci. 2022, 12, 2782 10 of 12 responding quantum yield of AcTDA was about 13% higher than that of TDA. In addition to the increased glucose binding affinity, the introduced acetyl group enhanced glucose responding quantum yield. Figure 6. Gradient plot of integrated fluorescence intensity against absorbance for (a) AcTDA and Figure 6. Gradient plot of integrated fluorescence intensity against absorbance for (a) AcTDA and fluorescein and (b) TDA and fluorescein. fluorescein and (b) TDA and fluorescein. 3.5. Interference-Dependent Fluorescence To use ForAcTD the A dr as a g ug libupr ucose probe, it ofen, nee AcTDA ds to demons did trat not e high show specifa icitmeaningful y for glucose fluorescence increase in over other interferences, such as six-carbon sugars and common drugs. Therefore, we the presence of maltose, sucrose, lactose, or ibuprofen (data not shown). Figure 7 shows measured the fluorescence changes of AcTDA with six different saccharides, including that the fluorescence increased when AcTDA was in the presence of fructose, galactose, fructose, galactose, lactose, maltose, mannitol, and sucrose, and the most common drugs. For the drug ibuprofen, AcTDA did not show a meaningful fluorescence increase in and mannitol. The association constants for fructose and galactose were determined the presence of maltose, sucrose, lactose, or ibuprofen (data not shown). Figure 7 shows using the modified Benesi–Hildebrand plots (Figure S2). However, the modified Benesi– that the fluorescence increased when AcTDA was in the presence of fructose, galactose, Hildebrand plot for AcTDA and mannitol did not exhibit a good fit (Figure S2). Therefore, and mannitol. The association constants for fructose and galactose were determined using the modified Benesi–Hildebrand plots (Figure S2). However, the modified Benesi– we derived a plot to fit the fluorescence changes in response to mannitol (Figure S3) and Hildebrand plot for AcTDA and mannitol did not exhibit a good fit (Figure S2). Therefore, obtained 213 4.5 M as the association constant. The association constants decreased in we derived a plot to fit the fluorescence changes in response to mannitol (Figure S3) and 1 1 −1 obtained 213 ± 4.5 M as the association constant. The association constants decreased in magnitude in the following order: mannitol (213 4.5 M ) > fructose (164 3.1 M ) > −1 −1 magnitude in the following order 1 : mannitol (213 ± 4.5 M ) > fructose (164 ± 3.1 M ) > galactose (55 6.1 M ). The association constant for glucose was at least 3.6-fold higher −1 galactose (55 ± 6.1 M ). The association constant for glucose was at least 3.6-fold higher than those of the interfering saccharides. As the blood interference level (<0.1 mM) is much than those of the interfering saccharides. As the blood interference level (<0.1 mM) is much lower than that of glucose (3.6−5.8 mM), the quantity of glucose-bound AcTDA would be lower than that of glucose (3.6 5.8 mM), the quantity of glucose-bound AcTDA would be 123-fold higher than that of the interference-bound AcTDA [31]. Therefore, the 123-fold higher than that of the interference-bound AcTDA [31]. Therefore, the interference Appl. Sci. 2022, 12, x FOR PEER REVIEW interferen ce could be considered negligible. 11 of 13 could be considered negligible. Figure 7. Binding properties of AcTDA with interferences. (a,c,e) Emission spectra for AcTDA with Figure 7. Binding properties of AcTDA with interferences. (a,c,e) Emission spectra for AcTDA with fructose, galactose, and mannitol, respectively. (b,d,f) Relative fluorescence intensity with fructose, galactose, and mannitol, respectively. fructose, galactose, and mannitol, respectively. (b,d,f) Relative fluorescence intensity with fructose, galactose, and mannitol, respectively. 4. Conclusions In conclusion, we successfully synthesized an oxidation resistant bis-phenyl boronate derivative with a large Stokes shift and long excitation and emission wavelengths by adding an acetyl moiety to the anthracene ring. The Stokes shift was the largest among the reported diboronic anthracene dyes. Moreover, the excitation wavelength was 420 nm, which was the longest of the reported diboronic anthracene dyes. The large Stokes shift and long excitation wavelength would allow the easy selection of an excitation-detecting unit and in vivo glucose monitoring. Moreover, the acetylated diboronic acid anthracene demonstrated good binding affinity and selectivity for glucose and an identical resistance to oxidation compared to the non-acetylated analog. The improved optical and glucose- detecting properties of the newly synthesized acetylated trifluoromethyl diboronic anthracenes can allow easy fabrication and reduce the power consumption of the system. Appl. Sci. 2022, 12, 2782 11 of 12 4. Conclusions In conclusion, we successfully synthesized an oxidation resistant bis-phenyl boronate derivative with a large Stokes shift and long excitation and emission wavelengths by adding an acetyl moiety to the anthracene ring. The Stokes shift was the largest among the reported diboronic anthracene dyes. Moreover, the excitation wavelength was 420 nm, which was the longest of the reported diboronic anthracene dyes. The large Stokes shift and long excitation wavelength would allow the easy selection of an excitation-detecting unit and in vivo glucose monitoring. Moreover, the acetylated diboronic acid anthracene demonstrated good binding affinity and selectivity for glucose and an identical resistance to oxidation compared to the non-acetylated analog. The improved optical and glucose-detecting properties of the newly synthesized acetylated trifluoromethyl diboronic anthracenes can allow easy fabrication and reduce the power consumption of the system. Therefore, we expect that the reported dye in this work will be utilized to develop an affordable and long-term usable implantable CGM system for widespread use. Supplementary Materials: The followings are available online at https://www.mdpi.com/article/ 10.3390/app12062782/s1. Figure S1: Excitation spectra in the presence of glucose for AcTDA (a) and TDA (b). Figure S2: The modified Benesi–Hildebrand plots of AcTDA with fructose (a), galactose (b), and mannitol (c), Figure S3: Association constant of AcTDA for mannitol. Author Contributions: H.C. designed and synthesized the dyes; I.S. and J.H.K. carried out the characterization of the dyes; C.S.P. worked on the data presentation; H.-s.Y. and J.H.K. conceived these studies. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by a National Research Foundation of Korea grant funded by the Korean government (NRF-2019M3E5D1A02068242). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. 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