DESIGN, SYNTHESIS OF A NEW CLASS OF DISSYMMETRIC MACROCYCLES FOR CARCINOGENIC BENZO[a]PYRENE DETECTION

Since 1987, synthetic macrocycles have gained much attention in supramolecular chemistry, especially for their use in the extraction and/or detection of specific guests. The binding of a guest within the host leads to the formation of a host-guest complex. These host-guest complexes are governed by a variety of non-covalent interactions such as π-π stacking, electrostatic interactions, Van der Waals forces, and hydrophobic interactions. Herein we report the rational design and synthesis of a series of macrocycles as hosts for the evaluation of binding and detection of carcinogenic polycyclic aromatic hydrocarbons including benzo[a]pyrene. Benzo[a]pyrene is one of the most carcinogenic, mutagenic and teratogenic polycyclic aromatic hydrocarbons and persists in the environment ubiquitously. Current detection methods involve tedious procedures and require multiple instruments for analysis. Hence, there is a need to find more efficient detection methods for this carcinogenic benzo[a]pyrene. The synthesized macrocycle hosts were evaluated for the efficient binding of benzo[a]pyrene and a high quantum yield fluorophore in the cavity of the macrocycle to generate ternary complexes. Proximity-induced energy transfer from the benzo[a]pyrene to a fluorophore resulted in a bright, turn-on fluorescence signal that can be used for benzo[a]pyrene detection. These complex systems also provide a key information about the intermolecular interactions that are required for efficient energy transfer to occur, including hydrophobic binding and π-π stacking. While synthesizing these macrocycles, we explored the development of new organic reactions such as green bromination of benzylic alcohols to their benzylic bromides, to optimize and complete the macrocyclization reaction and minimize the generation of environmentally toxic waste products. We have also explored highly efficient and sensitive detection methods for cesium metal ion in aqueous media and for hydrogen peroxide, both in solution and vapor phase. The first manuscript,“Highly efficient non-covalent energy transfer in all-organic macrocycles,” focuses on the use of aromatic organic macrocycles as supramolecular hosts for non-covalent energy transfer. These macrocycles lead to stronger binding and more efficient energy transfer compared to commercially available γ-cyclodextrin. This energy transfer was particularly efficient for the highly toxic benzo[a]pyrene with a fluorescent BODIPY acceptor, with up to a 5-fold increase in the fluorophore emission observed. The second manuscript,“A series of dissymmetric macrocycle hosts for the facilitated detection of carcinogenic benzo[a]pyrene,” describes a series of electronically dissymmetric organic macrocycles that were synthesized and evaluated for the facilitated efficient detection of highly toxic and carcinogenic benzo[a]pyrene via non-covalent energy transfer. This proximity-induced energy transfer was performed using a fluorescent BODIPY dye as an energy acceptor in combination with benzo[a]pyrene as the energy donor. Up to a 300% increase in the resulting fluorophore emission from analyte excitation compared to the emission from direct excitation was observed in the presence of the macrocycle hosts. The third manuscript,“A green bromination method for the synthesis of benzylic dibromides,” describes the development of new methodology for the dibromination of benzylic diols. This method proceeds in moderate to good yields for a wide variety of electron-deficient, electronneutral, and electron-rich aromatic substrates. Moreover, the reagent, 1,3-dibromo-5,5dimethylhydantoin, and the solvent, tetrahydrofuran, are substantially more environmentally benign than traditional solvents and reagents used for bromination. The utility of this methodology was demonstrated in the high-yielding synthesis of a key intermediate in the synthesis of omeprazole. The fourth manuscript, “Sensitive and selective detection of cesium via fluorescence quenching,” describes the selective detection of cesium metal ion. Herein we report a robust and easy method for detecting cesium metal ion (Cs + ) in partially aqueous solutions using the fluorescence quenching of 2,4-bis[4-(N,N-dihydroxyethylamino)phenyl]squaraine. This squaraine dye was found to be both highly sensitive (low limits of detection) and selective (limited response to other metals) for cesium ion detection. The detection is likely based on the metal complexing to the dihydroxyethanolamine moieties, which disrupts the donoracceptor-donor architecture and leads to efficient quenching. The fifth manuscript, “Highly efficient detection of hydrogen peroxide in solution and in the vapor phase via fluorescence quenching,” describes a highly efficient and sensitive detection of hydrogen peroxide in both aqueous solution and in the vapor phase via fluorescence quenching (turn-off mechanism) of the amplified fluorescent conjugated polymer-titanium complex induced by hydrogen peroxide. Interand intra-polymer energy migration leads to extremely high sensitivity and substantial improvements compared to current state of the art methods.

Copies of all NMR spectra……………………………………………………………………30   Highly efficient non-covalent energy transfer in all-organic macrocycles Abstract: The use of aromatic organic macrocycles as supramolecular hosts for non-covalent energy transfer is reported herein. These macrocycles lead to stronger binding and more efficient energy transfer compared to commercially available γ-cyclodextrin. This energy transfer was particularly efficient for the highly toxic benzo[a]pyrene with a fluorescent BODIPY acceptor, with up to a 5-fold increase in the fluorophore emission observed.
The complexation of small molecules in organic macrocyclesis a highly active area of research, with applications including supramolecular catalysis, 1 small-molecule detection, 2 and macrocycle-promoted energy transfer. 3 We have previously shown that γ-cyclodextrin, a wellknown supramolecular host, 4 promotes efficient energy transfer from several polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) to fluorophore acceptors. 5 This energy transfer occurs with up to 160% efficiency, and has significant potential applications in developing array-based detection schemes. 6 The use of aromatic macrocycles as supramolecular hosts can lead to even stronger binding of aromatic guests and higher energy transfer efficiencies, as these macrocycles can bind aromatic guests via π-π stacking 7 in addition to hydrophobic binding. 8 Four examples of such macrocycles were synthesized ( Figure 1) (synthetic details provided in the ESI). Briefly, a double Williamson etherification reaction 9 followed by a double Suzuki reaction 10 rapidly assembled the linear precursors. The key macrocyclization reactions were accomplished via a double etherification reaction (for compound 1) 11 or via a double Mitsunobu reaction (for compounds 2-4). 12 3 These macrocycles include three structures that are electronically-dissymmetric (1)(2)(3), with clearly defined electron-rich and electron-deficient components to the macrocycle, and one that is electronically symmetric. The electronically dissymmetric structures are designed to bind an electron-rich analyte near the electron-deficient component of the macrocycle, and an electron-deficient fluorophore near the electron-rich segment of the macrocycle, to form a stack of four aromatic components with alternating electronic character that will undergo efficient energy transfer. Whether such dissymmetry improves the binding and energy transfer efficiencies was tested by comparison to control macrocycle 4, which lacks such dissymmetry.
Semi-empirical PM3-level calculations of the macrocycles indicate that all of them have internal dimensions analogous to that of γ-cyclodextrin (Table 1), 13 and sufficiently large to promote intra-cavity energy transfer.   Fitting this data to a Benesi-Hildebrand equation for a 1:2 complex revealed an apparent binding constant of 5 x 10 9 M -2 , 15 which is among the highest binding constants observed for this highly toxic analyte. 16 By comparison, the addition of macrocycle 2 to a solution of anthracene resulted in no significant changes in the anthracene emission beyond spectral broadening ( Figure 3b). This binding was further confirmed by 1 H NMR titration studies. 17 The titration of benzo[a]pyrene into a solution of macrocycle 2 in CDCl 3 resulted in a shift of both the benzo[a]pyrene peaks and the macrocycle peaks (Table 2; Figure 4). The fact that macrocycle protons C and D shift noticeably indicates that benzo[a]pyrene associates with both sides of the macrocycle, although more with the electron-deficient side (as indicated by a larger shift in the C protons). The simultaneous shifts in the host and guest peaks suggest a close association between the host and the guest, and are consistent with the fluorescence data.
6 In addition to their ability to bind PAHs, macrocycles 1-4 were also investigated for their ability to promote energy transfer from analytes 5 and 6 to highly fluorescent BODIPY 7. 18 The efficiency of such energy transfer was quantified in two ways: (a) by measuring the decrease in the donor emission from adding an energy acceptor, according to Equation 1: Where I DA is the integrated emission of the fluorophore from analyte excitation, and I A is the integrated emission of the fluorophore (from excitation at the same wavelength) in the absence of the analyte. The results of macrocycle-promoted energy transfer are summarized in Table 3. These experiments were conducted under mostly aqueous conditions to maximize the favourable hydrophobic binding and π-π stacking between the aromatic PAH donor, aromatic fluorophore acceptor, and aromatic macrocycle. The results clearly indicate that macrocycle 2 was the most efficient host for non-covalent energy transfer, as measured both by the increase in fluorophore emission more than 5-fold and by the decrease in donor emission to 57% of its initial value (Figure 5a and 5b). The minimal amount of excimer emission observed in these spectra strongly suggests that fluorophore 7 displaces one molecule of benzo[a]pyrene from the macrocycle's interior.
The only difference between the two hosts is the replacement of the perfluorophenyl ring in macrocycle 2 with a phenyl ring in macrocycle 4, which effectively removes the electronic dissymmetry from the structure. This direct comparison indicates that electronic dissymmetry provides a direct benefit for supramolecular energy transfer efficiencies.
Macrocycle 2 was also substantially more efficient at promoting such energy transfer compared to γ-cyclodextrin. 5  The reasons why macrocycle 2 is substantially more efficient than macrocycles 1, 3,and 4 at binding PAHs and promoting energy transfer are currently under investigation, but the following conclusions can already be drawn: (a) Electronic dissymmetry in the host leads to more efficient energy transfer (by comparing macrocycle 2 and 4); (b) the presence of an ester linkage leads to higher energy transfer efficiencies (by comparing macrocycles1 and 2); and (c) the presence of methoxy groups, which increases the electron-donating nature of the top half of the macrocycle, leads to less efficient energy transfer, perhaps by increasing the steric hindrance and limiting cavity accessibility. 9 Figure 5: Comparison of the energy transfer in macrocycle 2 (5a and 5b) and macrocycle 4 (5c and 5d).
In summary, reported herein is the use of aromatic organic macrocycles as supramolecular hosts for PAH binding and non-covalent energy transfer. One of the new macrocycles, compound 2, is substantially more efficient than known macromolecules at binding benzo[a]pyrene and promoting energy transfer from this toxicant to a fluorophore. More generally, the ability to modify the supramolecular host for this energy transfer via synthetic organic chemistry provides optimal flexibility in tuning and optimizing such non-covalent energy transfer. The scope of macrocycle-promoted energy transfer and its use in array-based detection scheme is currently under investigation. Compound S1 (1.50 g, 5.68 mmol, 1.0 eq.) and 3-bromobenzyl alcohol S2 (2.34 g, 12.50 mmol, 2.2 eq.) were dissolved in 20 mL of anhydrous dimethylformamide (DMF). The reaction mixture was stirred for 10 minutes at room temperature and was cooled to 0 o C in an ice bath. Sodium hydride (0.368 g, 15.34 mmol, 2.7 eq.) (60% in mineral oil) was then added to the reaction mixture and the reaction mixture was allowed to warm to room temperature.
14 The reaction mixture was stirred at room temperature for 2 hours, at which point it was determined to be complete by TLC. Distilled water (10 mL) was added to quench the reaction, and the reaction mixture was then extracted with 3 portions of dichloromethane (20 mL each time). The combined organic extract was washed 9 times with distilled water (20 mL each time), followed by washing with brine. The organic extract was then dried over sodium sulfate, filtered, and concentrated via rotary evaporation to yield the crude product. The product was purified by flash chromatography Reaction 8: Synthesis of compound S12: Compound S11 (1.0 g, 3.09 mmol, 1.0 eq.) and compound S2 (1.26 g, 6.74 mmol, 2.2 eq.) were dissolved in 10 mL of anhydrous dimethylformamide. The reaction mixture was stirred for 10 minutes at room temperature and was cooled to 0 o C in an ice bath. Sodium hydride (0.200 g, 8.33 mmol, 2.7 eq.) (60% in mineral oil) was then added to the reaction mixture and the mixture was allowed to warm to room temperature. The reaction mixture was stirred at room temperature for 2 hours, until it was determined to be complete by TLC. Excess distilled water (~15 mL) was added to quench the reaction, and the reaction mixture was then extracted multiple times with dichloromethane (3 x 20 mL). The combined organic extract was washed 10 times with distilled water (10 mL each time), followed by brine. The organic extract was then dried over sodium sulfate, filtered, and concentrated via rotary evaporation to yield the crude product. The crude product was purified by isopropanol (0.5 mL) and hexanes (10 mL) to afford the ether product S12 as a white solid in 62% isolated yield (1.02 grams) R f : 0.56
The reaction mixture was stirred for 2.5 hours at room temperature and then the reaction mixture was diluted with diethyl ether (10 mL). The organic phase was washed with saturated NaHCO 3 (2x10 mL) and brine (10 mL

Synthesis of BODIPY 7:
The synthesis of BODIPY 7 was performed according to literature procedures: was then added and the reaction mixture was stirred at 80 o C for 30 minutes, during which time the color of the mixture darkened and became fluorescent. The reaction mixture was cooled to room temperature, and the product was extracted 3 times with brine (50 mL each time). The organic layer was dried over sodium sulfate, filtered, and concentrated. The crude product was purified by flash chromatography (1:1 dichloromethane: hexanes) to yield the desired product S19 in 28% yield (comparable to the literature-reported 24% yield).

Reaction 2:
Procedure: Compound S19 (0.968 g, 2.07 mmol, 1.0 eq.) and compound S20 (0.27 grams, 2.36 mmol, 1.14 eq.) were dissolved in 50 mL of acetone. The reaction mixture was heated to reflux for two hours. After two hours, the reaction mixture was cooled to room temperature, 26 acetone was removed, and the crude solid was re-dissolved in dichloromethane and washed with water. The organic extract was dried over sodium sulfate, filtered and concentrated, to yield compound S21 in 97% yield (0.932 grams).

Reaction 3:
Procedure: Compound S21 (0.932 grams, 2.01 mmol, 1.0 eq.) was dissolved in 150 mL of anhydrous ethanol that was purged with nitrogen. Potassium carbonate was added, and the reaction mixture was warmed to 30 o C. The reaction mixture was stirred under nitrogen for 4 hours at 30 o C. The contents of the flask were poured over 40 mL of aqueous saturated ammonium chloride, at which point the solution turned bright orange. The product was extracted with dichloromethane and washed several times with water. The organic layer was dried over sodium sulfate, filtered, and concentrated. The product was purified via flash chromatography (1:1 dichloromethane: hexanes) to yield compound 7 in 76% yield (674 mg).

Synthesis of control BODIPY (compound S22):
27 All fluorescence spectra were recorded on a Shimadzu RF 5301 spectrophotometer. Binding experiments were conducted as follows: The following stock solutions were made: inside the cavity of the host, the phenomenon is termed "encapsulation." These host-guest complexes are governed by a variety of non-covalent interactions such as π-π stacking, electrostatic, Van der Waals forces, and hydrophobic interactions. [3][4][5] Supramolecular hosts including cyclodextrins, 6-9 calix[n]arenes, 10 Exbox, 11 and metallomacrocycles, 12,13 have been used in a variety of applications, including the extraction and detection of carcinogenic and highly toxic polycyclic aromatic hydrocarbons (PAHs).
PAHs represent an important group of ubiquitous environmental pollutants. 14,15 They are formed as byproducts from the incomplete combustion of fuel or organic materials, and have been found in the environment following oil spills. 16 Among all the PAHs, benzo[a]pyrene 8 was found to be one of the most carcinogenic, mutagenic and teratogenic. 17 Herein we report a substantial extension of this work, to include the synthesis of a series of dissymmetric organic macrocycles (  Computational studies using semi-empirical PM3 level calculations were performed to determine the dimensions of macrocycles 1-6. The calculated dimensions are analogous to the 47 reported cavity dimensions of γ-cyclodextrin (Table 1), 23 which has been used previously for PAH isolation and detection. 7-9, 21, 22  Where I DA is the integration of the fluorophore from analyte excitation and I A is the integrated fluorophore emission from direct excitation.
52 The energy transfer efficiency results of both macrocycles 1 and 5 are 322% and 345%, respectively and clearly indicate that they are the most efficient supramolecular hosts for facilitating non-covalent energy transfer (Table 2).   To truly assess the energy transfer efficiency, we compared the energy transfer efficiency of macrocycle 5 with one of the most efficient macrocycle 2 of our previous paper. 20 We observed that slightly less efficient energy transfer occurred in macrocycle 5. This could be due to less proximity between benzo[a]pyrene and BODIPY; current efforts are focused on elucidating the reasons for this difference. We have also calculated the limit of detection for benzo[a]pyrene using benzo[a]pyrene to BODIPY energy transfer in the presence of macrocycle 5, and found it to be 1.00 mM (see ESI for details).
Macrocycle 1 and 5 both have shown their efficiency in promoting energy transfer compared to our previously reported work with γ-cyclodextrin. 7,8 The reason macrocycles 2, 3, 4 and 6 are not efficient in promoting the energy transfer is most likely due to their inability to bind benzo[a]pyrene 8 efficiently, which in turn limits their ability to promote efficient energy transfer. Computational work also highlights the extent to which small structural changes in the macrocycles lead to significant changes in their cavity dimensions and their ability to form host-guest complexes with small molecule guests. Macrocycles 1 ( Figure 8A) and 5 ( Figure   8C) looks like rectangular in their cavity sizes whereas the introduction of methoxy substituents in the macrocycle 2 changed it's conformation ( Figure 8B) in such a way not to bind the analyte effectively and removal of methoxy groups in the macrocycle 6 ( Figure 8D) leads to changes its conformation not to bind the analyte efficiently.

Synthesis of Macrocycle 3:
To compound S5 (50.0 mg, 0.094 mmol, 1.0 eq.), 5-nitroisophthalic acid S12 ( The reaction mixture was allowed to warm to room temperature, and was stirred for 4 hours at room temperature. The reaction mixture was diluted with ethyl acetate (15 mL), and washed with saturated NaHCO 3 (2 x 10 mL portions) and brine (10 mL

Synthesis of BODIPY 7 fluorophore:
The synthesis of BODIPY 7 was performed according to literature procedures: cooled to room temperature, and the product was extracted 3 times with brine (50 mL each time). The organic layer was dried over sodium sulfate, filtered, and concentrated. The crude product was purified by flash chromatography (1:1 dichloromethane: hexanes) to yield the desired product S18 in 28% yield (comparable to the literature-reported 24% yield).

Reaction 2:
Procedure: Compound S18 (0.968 g, 2.07 mmol, 1.0 eq.) and compound S19 (0.27 grams, 2.36 mmol, 1.14 eq.) were dissolved in 50 mL of acetone. The reaction mixture was heated to reflux for two hours. After two hours, the reaction mixture was cooled to room temperature, acetone was removed, and the crude solid was re-dissolved in dichloromethane and washed with water. The organic extract was dried over sodium sulfate, filtered and concentrated, to yield compound S20 in 97% yield (0.932 grams).

Reaction 3:
Procedure: Compound S20 (0.932 grams, 2.01 mmol, 1.0 eq.) was dissolved in 150 mL of anhydrous ethanol that was purged with nitrogen. Potassium carbonate was added, and the

Benesi-Hildebrand Plots for all Macrocycle-Benzo[a]pyrene Combinations:
Benesi-Hildebrand plots for Macrocycle 1 and 5 are included in the main text.    The following procedure was used to determine the limit of detection and limit of quantification for each fluorophore-analyte combination.
We prepared the following solutions: 5. For each case, the fluorophore with macrocycle (before any analyte was added), was also excited at the excitation wavelength for the analyte, and the fluorescence emission spectrum was recorded (as per step 1). These measurements are referred as the "blank." 6. The limit of the blank is defined according to the following equation:

LoB LOD = m blank + 3(SD blank )
Where m is the mean of the blank integrations and SD is the standard deviation.
7. The limit of the blank was then entered into the equation determined in step 4 (for the y value), and the corresponding X value was determined. This value provided the LOD in mM 8. The limit of quantification (LOQ) was calculated in a similar way to the limit of detection.
First, the limit of the blank for quantification was determined according to the following equation: LoB LOQ = m blank + 10(SD blank )

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This value was then entered into the equation determined in step 4 (for the y value), and the corresponding X value was determined. This value provided the LOQ in mM.

Introduction
The bromination of benzylic alcohols to yield benzylic bromides is a widely used transformation in synthetic organic chemistry, 1 with applications in the synthesis of key drug intermediates, 2 natural products, 3 highly functionalized materials, 4 and multiple dyes and pigments. 5 Conventional reagents for the bromination of benzylic alcohols include molecular bromine, 6 hydrobromic acid, 7 carbon tetrabromide, 8 and N-bromosuccinimide (NBS). 9 Conventional solvents for this transformation include chloroform, dichloromethane, and carbon tetrachloride.
All of the brominating reagents and solvents listed above have been shown to be harmful to the environment, 10 toxic to a wide variety of organisms, 11 and expensive to use and dispose of safely. 12 More environmentally benign reagents and solvents that efficiently brominate a wide variety of substrates would provide significant operational advantages in accomplishing such synthetic transformations while limiting the potential environmental damage.
Some examples of environmentally benign bromination methods include the use of solventfree conditions, 13 ionic liquids, 14 and aqueous solvents 15 to promote the reactions of organic substrates with bromine-containing salts. The substrates for these reactions include both alkenes and aromatic compounds; however, bromination of benzylic alcohols using environmentally benign reagents has not been reported to date.
1,3-dibromo-5,5-dimethylhydantoin (DBDMH, compound 2) has been well-studied in the literature as a catalyst, 16 oxidant, 17 and commercial disinfectant. 18 It has also been used as a bromination reagent for aromatic C-H bonds, 19 alkenes, 20 and alkynes. 21 These literature precedents prompted our investigation into the use of this reagent as a less toxic bromination reagent to achieve efficient benzylic bromination.

Results
Initial investigations focused on the synthesis of dibromide 3a, driven by ongoing research in the synthesis of electronically-differentiated macrocycles (Scheme 1). 22 23 and led to only a mild reduction in the product yield.

Scheme 1. Synthesis of 3a
Under these optimized conditions, a wide variety of electron-deficient benzylic diols were converted to their corresponding dibromides in moderate to good yields (Scheme 2, compounds 3a-3g). Multiple substitution patterns were well-tolerated (both 1,4-diols and 1,3diols worked well), as were a wide variety of electron-deficient substituents. Interestingly, whereas one bromine substituent was well-tolerated (compound 3c), the introduction of two bromine substituents completely shut down the bromination to form compound 3f. Rather, a mixture of mono-and dialdehydes was formed under these conditions. DBDMH is a wellknown oxidant; 17 however, it is interesting that this is the only substrate for which such reactivity was observed. These reaction conditions also worked well for a variety of electron-neutral and electronrich benzylic diols (compounds 3h-3n), which formed the benzylic dibromide products.
Again, multiple substitution patterns were well-tolerated, with 1,2-, 1,3-and 1,4-diol substrates proceeding in high yields. The only limitation observed for these substrates is that the presence of a hydroxyl group or methoxy group at the meta position led to a 0% yield of compounds 3j and 3k. Instead, substrates 1j and 1k underwent both benzylic bromination as well as aromatic bromination to form compounds 3jj and 3kk, in accordance with the literature precedent (see ESI for a detailed structural elucidation). 24 The strongly activating nature of the hydroxyl group, combined with its small steric size, leads to the bromination of all available ortho-and para-positions to form 3jj.The methoxy substituent, by contrast, directs para-bromination to form 3kk, but has sufficient steric bulk to shut down the orthobromination pathway. The bromination reaction also proceeded well for a heteroaromatic diol to yield the desired dibromide in moderate yield (compound 3n).  Table 2, and indicate that the DBDMH bromination yields were equal to or higher than yields obtained using CBr 4 for all substrates investigated. Whereas CBr 4 worked well for electrondeficient substrates 1a-e, it was much less efficient for electron-rich substrates. DBDMH, by contrast, led to moderate to good bromination yields in all cases, and has the added advantage of being substantially less toxic to both the environment and to human health. Although higher yields for DBDMH-promoted reactions could likely be found in chlorinated solvents, our focus on green chemistry led us away from pursuing that direction of research.

Discussion
The proposed mechanism of this reaction is shown in Scheme 3, and involves the initial formation of a phosphonium bromide salt 5 and highly resonance-stabilized anion 6, which deprotonates one of the benzylic diols to form anion 8. Nucleophilic attack of compound 8 on

Conclusion
In conclusion, a new methodology for benzylic bromination using an environmentally friendly solvent and reagent is reported herein. This methodology has a number of advantages compared with traditional bromination reactions, including the ability to achieve good yields for a wide range of electron-rich, electron-deficient, and electron-neutral substrates, the substantially reduced reagent toxicity, and the ability to conduct these reactions in environmentally benign tetrahydrofuran rather than more toxic chlorinated solvents. The applications of this methodology in the synthesis of more complex molecules, as well as detailed mechanistic investigations, are currently underway in our group, and results will be reported in due course.

MATERIALS AND METHODS
All reactions were carried out under a dry nitrogen atmosphere unless otherwise noted.
Solvents were dried using an MBraun dual solvent purification system prior to use.

Synthesis of 2,5-bis(hydroxymethyl)bromobenzene (1c):
Compound S4 was prepared according to the reported procedure. 2 Synthesis of 2,5-bis(hydroxymethyl)bromobenzene (1c): To a stirred solution of S4(1.24 g, 4.54mmol, 1.0 eq.)in anhydroustetrahydrofuran (35 mL) at 0 o C was added lithium aluminum hydride (0.43 g, 11.35 mmol, 2.5 eq.)portion-wise slowly over a period of 5 minutes. After the addition was complete, the reaction mixture was stirred at 0 o C for 2.5 hours. After the completion of the reaction, the reaction mixture was quenched by the slow addition of methanol (3 mL), followed by the slow addition of water (5 mL

Synthesis of 3,5-bis(hydroxymethyl)phenol (1j):
A literature method was used to synthesize 3,5-bis(hydroxymethyl)phenol (1j). 5 To a THF (10 mL) suspension of LiAlH 4 (0.34 g, 8.88mmol, 3.7 eq.) was added a 5 mL THF solution of dimethyl 5-hydroxyisophthalate (S11) (0.50 g, 2.37 mmol, 1.0 eq.) dropwise at 0 o C. The reaction mixture was then refluxed for 6 hours. After completion of the reaction, a 10% aqueous H 2 SO 4 solution (0.5 mL) was carefully added dropwise at 0 o C until no more hydrogen evolved. The reaction mixture was filtered, and the filtrate was evaporated via rotary evaporator to obtain the product 1j as a white solid (147 mg, 40% yield). 1   (758 mg, 2.89 mmol, 2.2 eq.) was then added and the reaction mixture was warmed to room temperature and stirred for 2 hours. The reaction progress was monitored by checking TLC.
After completion of the reaction, the THF was evaporated under reduced pressure. The crude product was purified by flash chromatography using hexanes:ethylacetate (9:1) to afford compound 3b as a red liquid (184 mg, 40% yield). 1
Triphenylphosphine (320 mg, 1.22 mmol, 2.2 eq.) was then added and the reaction mixture was warmed to room temperature and stirred for 2 hours. After completion of the reaction, THF was evaporated under reduced pressure. The crude product was purified by flash chromatography using hexanes: ethylacetate (9:1) to afford compound 3d as an off-white solid (68 mg, 40% yield). 1
for 2 hours. After completion of the reaction, THF was evaporated under reduced pressure.
After completion of the reaction, THF was evaporated under reduced pressure. The crude product was purified by flash chromatography using hexanes: ethylacetate (9:1) to afford compound 3h as an off-white solid (82 mg, 43% yield). 1
After completion of the reaction, THF was evaporated under reduced pressure. The crude product was purified by preparative thin layer chromatography plates using hexanes: ethylacetate
After completion of the reaction, THF was evaporated under reduced pressure and the crude reaction mixture was dissolved in ethylacetate (10 mL) and extracted with 1N HCl (2 x 5 mL).The aqueous HCl layer was basified with 1N NaOH (11 mL to reach pH 8

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The reactions of compounds 1j and 1k led to a 0% yield of the desired dibrominated products 3j and 3k. However, in both cases, a single major product was formed. We undertook a detailed structural elucidation of that product, as shown below: Elucidation of 3jj: Cesium, which is found in industrial, 1 medical 2 and nuclear wastes, 3 can cause a number of negative health effects, including cardiovascular disease and gastrointestinal distress. 4 Current methods for detecting cesium in complex environments include atomic absorption spectroscopy (AAS), 5 inductively coupled plasma mass spectroscopy (ICP-MS), 6 and solid state sensors. 7 While these methods are highly sensitive and selective for cesium detection, they are often expensive and require sample destruction.

2,4,6-tribromo-3,5-bis(bromomethyl)phenol
Fluorescence-based methods have rarely been used for cesium detection, 8 even though such methods have the potential to be both cheaper and non-destructive, 9 and have been used successfully for the many analytes. 10 In one example of fluorescence-based cesium detection, researchers synthesized a substituted calixarene that bound cesium with high affinities, resulting in a significant fluorescence enhancement and a 0.3 µM detection limit. 11 Reported herein is a highly sensitive and selective method for cesium detection that relies on the fluorescence quenching of a near-infrared emitting squaraine fluorophore (compound 1).
Squaraine fluorophores are used extensively in detection schemes due to their narrow absorption and emission bands in the near-infrared spectral region, as well as their marked sensitivity to the surrounding environment. 12 Squaraines have been used to detect metal ions, including mercury, silver, and lead, as well as other transition metals, alkali metals, and lanthanide metals. 13

Compound 1
Compound 1 was synthesized following literature-reported procedures. 14     The sensitivity of this method was determined by calculating the limit of detection. Although this limit is typically defined as three times the standard deviation of the background noise (or ten times the standard deviation for quantification limits), 15 in this case that definition led to a value that was effectively zero. Using 30 times the standard deviation of the background noise for these calculations led to a limit of detection of 0.096 µM, which provides an upper boundary for the detection limit. This detection method is more sensitive than previously reported fluorescence-based methods for purely aqueous solutions, 11 as well as for cesium ion detection in mixed solvent systems. 16 The selectivity of this detection method was determined by screening a wide variety of other metals, including transition metals, alkali metals, and alkali earth metals in a variety of oxidation states. Most of these ions led to no significant changes in the squaraine's fluorescence spectra (Figure 4). Preliminary experiments indicate that both the carbonate anion and cesium cation are necessary for the highly efficient fluorescence quenching, as no fluorescence quenching was observed for the following species: Cs 2 SO 4 , CsNO 3 and CsI. Both K 2 CO 3 and Na 2 CO 3 induced some degree of squaraine fluorescence quenching, albeit significantly less than the quenching observed for Cs 2 CO 3 (at 1.0 mM metal ion: 7.6%, 28% and 29% of initial fluorescence was observed for Cs 2 CO 3 , K 2 CO 3 and Na 2 CO 3 , respectively).  The observed fluorescence quenching is in line with a literature report of a crown-ether containing squaraine whose fluorescence was quenched in the presence of alkali and alkali earth metals. 17 In that report, the authors concluded that the metal ions caused fluorescence quenching through binding in the crown ether moieties, which disrupted the donor-acceptordonor nature of the squaraine chromophore. 18 Similarly, in this case we expect that the cesium cation binds strongly to diethanolamine, attenuating its strongly donating character and leading to highly efficient fluorescence quenching. 19

Conclusions
In conclusion, reported herein is a sensitive and selective method for detecting cesium via

Materials and Methods
All reagents and solvents were purchased from Sigma Aldrich and were used as received without further purification. 1 H NMR spectra were recorded on a Bruker 300 MHz spectrometer. Fluorescence measurements were recorded on Shimadzu RF 5301 spectrophotometer with a 3 nm excitation slit width and a 3 nm emission slit width. All spectra were integrated vs. wavenumber using OriginPro software.
Phenyldiethanolamine S1 (0.88 mmol, 160 mg, 2.0 eq.) and squaric acid S2 (0.44 mmol, 50 mg, 1.0 eq.) were dissolved in a 1:1 mixture of toluene and n-butanol. The reaction mixture was equipped with a Dean-Stark trap and condenser, and the reaction mixture was stirred in the dark at reflux overnight. The reaction mixture was cooled to room temperature and then to 0 o C. The precipitate was collected by vacuum filtration and thoroughly dried to yield 19 mg of compound 1 (12.5% yield).

Screen of Other Metals
All non-interacting metals were screened using the following procedure: All metal salts were dissolved in distilled water to a final concentration of 10 mM. Compound After thorough mixing, the fluorescence spectra were recorded from 650 nm excitation with 3 nm excitation slit width and 3 nm emission slit width.

Counterion Effect
The following additional metal salts were screened to probe the effect of the counterion:    The limit of the blank was taken to be the average of the blank (squaraine emission without cesium) + 30 times the standard deviation of the blank.
This value was entered into the equation determined in step 3 (for the Y value), and the corresponding X value was determined. This value provided the LOD in mM.

MANUSCRIPT 5
This manuscript is published in Chem. Commun., 2015,   The detection of hydrogen peroxide (HP) remains a crucial research objective, as hydrogen peroxide has been used in the manufacture of homemade explosives, 1 and has caused significant accidental explosions, even at low concentrations. 2 The presence of elevated levels of hydrogen peroxide in biofluids indicates significant oxidative stress; 3 such stress can cause long-term oxidative damage to cells and organs. 4 Despite the importance of detecting hydrogen peroxide in multiple environments, the reactive and transient nature of hydrogen peroxide means that it is difficult to develop direct detection methods. Most detection methods for hydrogen peroxide react the hydrogen peroxide with a substrate, and monitor the conversion of that substrate to product using a variety of analytical techniques, 5 including electrochemistry, 6 chemiluminescence, 7 and fluorescence spectroscopy. 8,9 Such indirect methods have been used successfully in a number of cases, including the hydrogen peroxide-induced hydrolysis of boronate esters, which often correlates with a detectable fluorescence change. 8 Colorimetric-based methods have also been 161 developed, wherein the introduction of hydrogen peroxide leads to a visible change in the color of the sensor that can be quantified to measure hydrogen peroxide concentrations. 10 In one reported example, titanium-oxo complex 1 was adsorbed on paper towels. 11 Upon exposure to the vapor of a 35 weight% solution of hydrogen peroxide, the paper towel turned from colorless to yellow due to the formation of titanium-oxo complex 2 (Scheme 1). 12 Most literature reports about liquid phase hydrogen peroxide detection via fluorescence enhancement as the basis for detection ("turn-on mechanism"); 8 meanwhile, there are a few studies where fluorescence quenching ("turn-off mechanism") has been employed as a transducer signal. 9 It was demonstrated in pioneering works by Swager's group that the fluorescence quenching of sensory conjugated polymers results in amplification of the responsive signal due to the energy migration effect. 13 The exciton energy migration along the polymer chain provides effective trapping and quenching of excitations generated by light, which is much greater than the quenching observed for isolated molecules (i.e. the concept of "amplifying polymers" (AMP) used in chemosensing). 14  Reported herein is the detection of hydrogen peroxide in both solution and in the vapor phase, using a combination of titanium complex 1, fluorescent conjugated polymer 3, and inert polymer 4(Chart 1).

Chart 1 Structures of compounds used for hydrogen peroxide detection
The introduction of hydrogen peroxide led to the highly efficient fluorescence quenching of polymer 3 in these complex mixtures, through energy migration along the polymer backbone.
The critical feature of this research is the application of AMPs to the design of an HP detection system, resulting in extraordinary sensitivity to HP vapors in the solid state (detection limit is ~ 200 ppt), which significantly exceeds previously reported results. [8][9][10][11] Fluorescence quenching-based detection methods have a number of advantages compared to other methods, 15 including the potential for high sensitivities, 16 rapid response times, 17 and straightforward experimental design and set-up. We have previously reported the use of fluorescence quenching for the detection of electron-deficient nitroaromatic compounds 18 and cesium carbonate. 19 The system reported herein has a number of notable advantages, including the use of a solid-state fluorescent film to detect extremely low vapor concentrations of hydrogen peroxide via highly efficient fluorescence quenching.
Polymer 3 was mixed with titanium complex 1 in two ways: by mixing the two compounds in an aqueous solution, and by co-depositing the two compounds on spin-cast thin films. In neither case were the polymer and titanium complex covalently linked; however, the electrostatic complementarity between the negatively charged polymer and positively charged 163 titanium complex enabled such association. This close association meant that the hydrogen peroxide-induced conversion of compound 1 to compound 2 directly influenced the fluorescence emission spectra of polymer 3, leading to highly efficient fluorescence quenching. The association between polymer 3 and complex 1can be confirmed by the fact that the absorption spectrum of solution of 3 and 1 is different from a sum of spectrum 1 and spectrum 3 ( Figure 1A).   Next, we corrected Stern-Volmer (SV) plots on the primary screening effect taking into account the optical densities of 2 and 3 for the three excitation wavelengths at increasing concentrations of compound 2 (see ESI). The dashed lines in Figure 3A show the calculated plots (eqn S5 in ESI) with the absence of the energy transfer (K ET = 0) and presents the contribution of the primary screening effect for each excitation wavelength (330 nm, 350 nm, and 370 nm). The slopes of these dependencies are significantly smaller than the slope of the 166 measured SV plots, which indicates an existence of the energy transfer effect. Figure 3B demonstrates the corrected SV plots obtained by dividing the measured SV plots by the calculated contributions of the primary screening effects for the three excitation wavelengths.
As it is expected after this correction, the SV plots at 350 and 370 nm excitation are almost identical, however they are slightly distinctive from the SV plot at 330 nm excitation. The latter fact can be associated with additional absorption of hydrogen peroxide itself at 330 nm ( Fig. 1A), which affects the correction term in Equation S5 (ESI). Considering the linear SV plots at 350 and 370 nm only, an energy transfer constant of K ET = 1180 M -1 can be deduced.
The most interesting results were obtained using thin films for the detection of HP vapors via fluorescence quenching. Attempts to directly spin-coat aqueous solutions of polymer 3 on thin films were unsuccessful, likely due to difficulties in fully evaporating water from the film 21 However, the addition of inert polymer 4 to the solution prior to thin film formation enabled the successful spin-coating of fluorescent thin films, by providing a hydrogen-bonding matrix for polymer 3. 22 The films were spun-coat from hot aqueous solutions of compounds 1, 3, and peroxide in the vapor phase (approximately 0.27 ppb and 2.7 ppb for the 30 ppm and 300 ppm solutions, respectively). 23 Thus, the detection limit (DL) for HP vapors can be estimated as low as ~ 200 ppt, which is significantly lower than limits reported by Sanchez et al (300 ppb) 8 and by Xu et al (400 ppb). 11 Such substantial improvements in the system's sensitivity to HP vapors is related to an amplifying polymers effect (turn-off mechanism), which outperforms colorimetric chemosensing or fluorescent sensory polymers with turn-on mechanisms. 8,10,11 We already noted that AMP provides an extremely high sensitivity due to energy migration along the polymer backbone, resulting in effective fluorescence quenching ("turn-off") when multiple excitations (excitons) can be quenched by a single analyte.
Meanwhile, energy migration through the polymer chain resulting in fluorescence enhancement ("turn-on" mechanism) is not so obvious as no direct evidence of the amplification effect has been presented in most studies, 24 (with few exceptions). 25 Turnoff AMPs, in contrast, have been confirmed by the comparison of the responsive signal between the monomer and polymer species. 13 Furthermore, films of amplifying polymers demonstrate increased sensitivity due to inter-polymer energy migration 26 compared to isolated polymer chains in solution or films composed from highly diluted polymers by inert matrix. We have found the same trends in quenching efficiencies for our system, with substantially increased sensitivity in thin films compared to in the solution state.  Table 1 shows how the fluorescence quenching (I o /I ratio after 9 min exposure to HP vapors) depends on varying concentration of polymer 4 (at constant concentration of 3 and 1) in the solutions prepared for spin coating (the inner filter effect is negligible because of the film's small thickness). As it can be seen, there is a clear trend of more efficient quenching with decreasing fractions of inert polymer 4, strongly implying the presence of inter-polymer energy migration between chains of polymer 3. Thus, the detection limit can be further reduced by approximately 40% using lower concentrations of polymer 4 ([4]=10g/L). It is noteworthy that optimization of the preparation methods for thin films (i.e. using dip coating or Langmuir-Blodgett techniques) could additionally improve the film sensitivity to the low parts per trillion range, due to more equilibrium deposition and improved polymer chains alignment. The proposed mechanism of fluorescence quenching likely involves the hydrogen peroxideinduced conversion of compound 1 to compound 2 (Scheme 1). Whereas the presence of compound 1 has no effect on the fluorescence of polymer 3, compound 2 acts as a strong fluorescence quencher using amplified fluorescence quenching. Figure 5shows a schematic illustration of possible mechanisms of fluorescence quenching upon HP exposure for isolated polymer chains (solution, highly diluted films) and for a polymer network (low diluted films).
Here the intra-and inter-energy migration mechanisms are presented by small blue arrows (exciton hopping between adjacent conjugated segments along the polymer chain) and green small arrows (exciton hopping between polymer chains in the junction area), respectively. Red arrows represent direct energy transfer from polymer 3 to complex 2. It is expected that at the limit of no dilution of polymer 3, that the sensitivity should be maximal and controlled by inter-and intra-3D energy migration through the densely packed chains of polymer 3 only.
Efforts to fully understand this quenching mechanism and to use these results to develop practical hydrogen peroxide sensors are in progress. The thin films were then cut in half and placed on a rubber septa that was inserted into a 1 cm 2 quartz cuvette ( Figure S1). The film's fluorescence was recorded for approximately 1 minute, and then an aqueous hydrogen peroxide solution was added to the cuvette via pipette, with the rubber septa ensuring that the thin film did not directly contact the hydrogen peroxide solution. The fluorescence spectra of the solution were recorded for several minutes after the hydrogen peroxide addition, to observe the solid-state fluorescence quenching in the presence of hydrogen peroxide vapors. All films were excited at 350 nm. The fluorescence declined to 48% of its initial value after 9 minutes.
Plotting this data as the change in I o /I over time:

SUMMARY FIGURES FOR THIN FILM QUENCHING EXPERIMENTS:
Varying the concentration of titanium complex 1: Change in I o /I vs. time: