Synthesis and Spectral Properties of 8-Anilinonaphthalene-1-sulfonic Acid (ANS) Derivatives Prepared by Microwave-Assisted Copper(0)-Catalyzed Ullmann Reaction.

For decades, the dye 8-anilino-1-naphthalenesulfonic acid (ANS) has been used to study biological systems due to its environmentally sensitive fluorescent nature and propensity to bind to hydrophobic pockets of proteins. However, the syntheses of ANS and its derivatives have been low yielding, requiring harsh reaction conditions and long reaction times. We have developed efficient, mild microwave-assisted copper(0)-catalyzed Ullmann coupling conditions to synthesize ANS derivatives with yields of up to 74%. Many of these derivatives have spectral properties distinct from ANS, including improved and diminished quantum yields, different absorption and emission maxima, and complete loss of fluorescence. ANS derivatives with these unique fluorescence properties are useful tools to study biological systems.

Introduction

(Phenylamino)naphthalenesulfonic acids have been used as dye intermediates since the 19th century.[1] Later, it was discovered that compounds like 8-anilino-1-naphthalenesulfonic acid (ANS; Scheme , 3a, R = H) were weakly fluorescent in water but had a much stronger fluorescence yield in the presence of less polar solvents and proteins with hydrophobic binding pockets.[2] ANS binds to proteins nonspecifically by anchoring to cationic side chains of proteins via its sulfonate moiety and additionally binding to nearby hydrophobic pockets on the protein via its anilinonaphthalene core.[3] The ability of ANS to bind hydrophobic pockets of proteins in this nonspecific manner has made it useful to study the properties of many biological systems. For example, ANS has been used to study proteins such as serum albumin,[4] apomyoglobin,[5] histone,[6] and alcohol dehydrogenase.[7] In conjunction with an increased fluorescence yield, less polar environments also cause a stark blue shift in the maximum emission wavelength of ANS.[8,9] The hypsochromic emission is due to a dramatically decreased Stoke shift in the nonpolar environment around polar, solvatochromic dyes.[9] The spectral properties of ANS and its derivatives have made these fluorophores useful for studying protein binding,[10] the protein “polarity” in enzymatic binding sites,[8] and action potentials in neuronal systems.[11] Recently, these fluorophores have been useful in studying protein aggregation in neurodegenerative diseases like Alzheimer’s and prion diseases.[12,13]

Scheme 1

Approaches to Synthesize ANS

Despite the utility of ANS and its derivatives to study important biological systems, the synthesis of these molecules has been challenging. Two approaches developed in previous decades (Scheme ) use sulfuric acid or aluminum trichloride as a catalyst at temperatures above 155 °C for longer than 10 h.[14−16] These approaches suffer from poor yields (less than 5%) and complicated product isolation. Therefore, it is necessary to develop an efficient approach for synthesizing ANS and its derivatives. In this work, we have used Ullmann coupling to develop a method for the synthesis of these compounds with improved yields and shorter reaction times. Ullmann coupling is a well-documented method to form C(aryl)–N bonds, like the one found in ANS.[17] This reaction has been used to synthesize 4-anilino-substituted anthraquinone derivatives, similar to ANS in its tolerance of the sulfonate group.[18−20] Upon synthesis, we subsequently characterized the fluorescence properties of our ANS derivatives.

Results and Discussion

We initially investigated two Ullman coupling conditions previously reported for anthraquinone derivatives (see Supporting Information).[18−22] Method A, which used elemental Cu as the catalyst and aqueous NaH2PO4 and Na2HPO4 solutions as the solvent, provided ANS (3a) in 45% yield. No product was obtained from method B, which involved CuI and tetramethylethylenediamine (TMEDA). Therefore, we optimized the Ullmann coupling conditions from Method A for the synthesis of ANS by screening copper catalysts, temperature, the amount of aniline, and reaction times. We screened copper catalysts CuI, CuCl, and Cu0, whereby Cu0 provided the best yield. Exploring a temperature range from 80 to 120 °C showed that 100 °C provided the best yield under microwave conditions. Investigation of the amount of aniline and reaction time indicated that 1.1 equiv of aniline and 1.5 h reaction time provided the best yield, with no benefit observed with more aniline or longer reaction times. The best reaction conditions combined 8-chloro-1-naphthalenesulfonic acid (1, 0.41 mmol, 1 equiv) with aniline (2a, 0.46 mmol, 1.1 equiv) in the presence of a catalytic amount of elemental copper (10 mol %). In 5 mL of aqueous sodium phosphate buffer of pH 6–7, the reaction mixture was stirred and microwaved for 1 h at 100 °C providing NAS (3a) in 63% yield. In order to investigate the application and tolerance to functional groups of this method, we next synthesized ANS derivatives with various aryl substitutions (Table ). Substrates with different halides on the benzene ring (3b–3h) resulted in good to excellent yields (42–67%). Not surprisingly, substrates with electron-donating groups such as methyl, methoxy, and acetylamino (3i, 3j, and 3o) gave the best yields of 69, 74, and 60%, respectively. Derivatives with electron-withdrawing groups, including nitrile and nitro, resulted in lower yields (3l–3m, 11–25%). Notably, tolerance of hydroxyl groups was observed (3k, 21%) in this reaction. The major side product of this approach is 8-hydroxynaphthalene-1-sulfonic acid caused by using water as a solvent, which is displacing the 8-chloro group.

Table 1

Synthesis of ANS Derivatives with Various Aryl Substitutionsa

products	R1	R2	R3	yields (%)b
3a	H	H	H	63
3b	H	H	F	64
3c	F	H	H	53
3d	H	F	H	47
3e	H	Cl	H	45
3f	H	H	Cl	62
3g	H	Cl	Cl	43
3h	H	H	Br	51
3i	H	H	Me	69
3j	H	H	OMe	74
3k	H	H	ONa	21
3l	H	H	CN	35c
3m	H	H	NO2	16c
3n	H	H	phenyl	23c
3o	H	H	acetylamino	60

Reagents and conditions: 1 (0.41 mmol, 1 equiv), 2 (0.46 mmol, 1.1 equiv), and Cu0 (10 mol %) are added into the buffer solution (pH 6–7) of Na2HPO4 and NaH2PO4 and irradiated by microwave for 1–1.5 h at 100 °C.

Isolated yields.

Reaction conditions were changed to 105 °C for 3 h.

The fluorescence properties of the synthesized ANS derivatives were analyzed. In particular, the maximum absorbance and emission wavelengths (λ) were recorded as well as the quantum yields (φ) in both water and the less polar solvent ethylene glycol, as reported in Table . Examples of absorbance and emission spectra are shown in Figure for 3f (the remaining absorbance and emission spectra can be found in the Supporting Information). Many ANS derivatives have been synthesized and characterized previously; our data closely matches what has been reported earlier.[16] As observed prior, less polar solvents resolve an absorbance spectrum with characteristic dual peaks, whereas in water, a single peak is observed. The maximum absorbance wavelength in ethylene glycol was recorded as the maximum of this second peak, as that peak confers maximal fluorescence for these compounds. All measurements were normalized to what has been reported and likewise observed in this study for the parent compound ANS, as is further described in the Experimental Section.

Table 2

Fluorescence Properties of ANS and Its Derivatives

cpds	λmax abs H2O (nm)	λmax abs ethylene glycol (nm)	λmax em H2O (nm)	λmax em ethylene glycol (nm)	φH2O	φethylene glycol	enhancing factor (φethylene glycol/φH2O)
3a	355	373	534	508	0.003	0.154	51.2
3b	347	362	553	507	0.001	0.040	60.3
3c	344	360	516	490	0.004	0.213	47.6
3d	345	364	509	482	0.009	0.441	50.4
3e	348	362	508	470	0.019	0.714	38.0
3f	355	370	536	507	0.003	0.099	28.6
3g	339	364	508	473	0.015	0.708	48.0
3h	356	371	533	499	0.005	0.127	26.8
3i	353	378	none observed	532	N/Aa	0.017	N/A
3j	350	375	none observed	562	N/A	0.003	N/A
3k	319b,c	351	none observed	508	N/A	0.008	N/A
3l	353	360	470	454	0.077	0.111	1.4
3m	418	420	none observed	none observed	N/A	N/A	N/A
3n	309b,c	322a	472	455	0.002	0.015	8.8
3o	360	380	none observed	558	N/A	0.004	N/A

N/A, not applicable.

Overlapping peaks.

Excited at 320 nm.

Figure 1

Representative example spectrum of 3f in water and ethylene glycol.

After much study and debate about the structural and energetic nature of ANS fluorescence, the model proposed by Kosower and Kanety has prevailed (Figure ).[16] In this model, the naphthalene and phenyl rings of ANS are noncoplanar (np) in the ground state (S0,np). Light excites an electron in the naphthalene ring without a significant change in configuration (S1,np). In less polar environments like ethylene glycol, a radiative pathway prevails over an alternative electron-transfer process that creates a conformationally different charged-transfer (ct) state between the two ring systems (S1,ct), which is stabilized in more polar environments such as in water. In polar environments, the S1,ct state can either nonradiatively relax to the ground state S0,np via another electron-transfer process or can emit light. In polar solvents, it has been well established that this nonradiative process is much faster than the radiative process, causing quenching of fluorescence signal.[23] Both solvents examined in this study, ethylene glycol and water, are relatively polar, allowing for rapid dissolution of the sodium salt ANS derivatives. However, notably, ethylene glycol is less polar and more viscous. Previous examination of ANS fluorescence in ethylene glycol and glycerol, solvents with similar polarities but substantially different viscosities, has demonstrated that while viscosity can somewhat influence the fluorescence properties of ANS and its derivatives, the polarity of the solvent is a more important determinant of these properties.[16]

Figure 2

Mechanism of fluorescence for ANS and ANS derivatives.

An enhancing factor was calculated, or the relative increase in fluorescence yield of the fluorophore upon change from a polar to less polar environment. This ideally correlates with the sensitivity range of the probe, or what difference in signal is expected if it were applied to a biological system. Four fluorophores (3i, 3j, 3k, and 3o) had an immeasurable fluorescence signal in aqueous environments that an enhancing factor could not be calculated. Such immeasurable fluorescence signals have been observed with similar ANS derivatives before.[24] Likewise, the fluorescence yields of these fluorophores were low even in ethylene glycol. The electron-donating substituent on all of these four derivatives likely better stabilized the S1,ct state and promotes the nonradiative relaxation pathway. One derivative, 3b (R1 = R2 = H, R3 = F), showed a slightly stronger enhancement in fluorescence than ANS when it transitioned from a polar to less polar environment. Four other derivatives (3c, 3d, 3e, and 3g) had a better fluorescence yield in ethylene glycol than ANS but additionally stronger background fluorescence in water. These four derivatives had electron-withdrawing halogen groups on the phenyl ring, which might not have stabilized the S1,ct state as well, pushing the equilibrium toward the S1,np state and radiative relaxation. Since ANS binds nonspecifically to hydrophobic pockets of proteins and only minor structural changes were made to these derivatives, these ANS derivatives might be better alternatives for those studying biological systems who want a more sensitive fluorescence probe or larger fluorescence signal upon binding to less polar local environments.

For many derivatives, the large blue shift of ANS upon exposure to a less polar environment is an attractive quality, as it further signifies a change in the chemical environment of the fluorophore. The derivatives synthesized in this study had a wide range of maximum absorption wavelengths in both water and ethylene glycol, from 310 to 380 nm. Likewise, there was an even wider range of maximum emission wavelengths in these media, from 454 to 562 nm. The energy corresponding to the maximum emission wavelength in ethylene glycol roughly corresponds to the Hammett substituent constant (see Supporting Information).[25] This trend has been observed for ANS derivatives previously.[16] The fluorescence yield, maximum emission wavelength, and enhancing factor are influenced by both the electronic nature of the aryl substituent and the local solvated environment. This gives biologists a new toolkit of fluorophores to use instead of ANS if different wavelengths are desired for either absorption or emission.

Lastly, one derivative, 3m (R1 = R2 = H, R3 = NO2) was not fluorescent in either solvent and had a much higher maximum absorption wavelength. Additionally, fluorescence emission was not observed up to 650 nm. These distinct properties likely arise from a significant resonance state where the electrons on lone pairs on the aniline nitrogen is conjugated with the electrons on the para nitro group, causing a substantially longer absorption wavelength and better stabilization of the S1,ct state by favoring a planar conformation. The conjugated nature of this derivative could give different excited states and/or relaxation pathways. While 3m absorbs energy, its inability to emit light via fluorescence makes it a suitable dummy probe for situations where fluorescence interference is a concern. The compound should still bind proteins, but not fluoresce.

Conclusion

ANS remains a useful fluorophore for studying biological systems. Traditionally, ANS derivatives with different spectral properties have been difficult to synthesize in high yields or under mild conditions. We have developed an efficient Ullmann coupling reaction for synthesizing ANS derivatives. This Ullmann coupling is unique in that it utilizes a chloride substituent instead of a bromide or iodide as the halide and is an example of a ligand-free Ullmann coupling. Additionally, our method adds to the literature of Ullmann coupling that tolerate a sulfonic acid moiety on the reactant; the sulfonic acid is a functional group usually synthesized last in a synthetic pathway as oxidation of a thiol group. Aside from the new application of the Ullmann coupling to the synthesis of these compounds, the ANS derivatives prepared in this study have a range of spectral properties distinct from ANS. These differences include improved and diminished quantum yields, different absorption and emission maxima, as well as compound 3m that does not fluoresce under conditions typical for ANS derivatives. Our method has allowed the incorporation of strong electron-withdrawing groups, such as the nitro group in 3m, whereas before, low yields have prevented the synthesis and analysis of such compounds. It is our hope that these new fluorophores provide biochemists with an expanded toolkit to better probe the most pertinent biological questions of today.

Experimental Section

General Materials and Methods

ANS derivatives were synthesized by the following procedure. 8-Chloronaphthalene-1-sulfonic acid (0.41 mmol, 1 equiv) and the substituted amine (0.46 mmol, 1.1 equiv) were placed into a 5 mL microwave reaction vial equipped with a magnetic stirring bar. A buffer solution of Na2HPO4 (pH 9.6, 4.5 mL) and NaH2PO4 (pH 4.2, 0.5 mL) was added to adjust the pH to 6.0–7.0 followed by the addition of a catalytic amount powdered elemental copper (10 mol %). The mixture was capped and microwaved (100 W) for 1–3 h at 100 °C. The products were purified by the following procedure. The pH of the reaction mixture was adjusted to above 12 by NaOH (5 M) followed by filtration to remove the elemental copper. Water (50 mL) was added to wash the filter residue. The aqueous layer was extracted with dichloromethane (40 mL, three times) to remove unreacted amine. Then, the aqueous layer was removed by rotary evaporation. The residue was dissolved in methanol (30 mL) followed by filtration to remove undissolved salts. The filtrate was concentrated by rotary evaporation, and the residue was subsequently purified by flash column chromatography using the reusable RediSep Rf Gold C18Aq column from Teledyne Isco (15.5 g and 30 mL/min flow rate) with MeOH/H2O (4:6). The average retention time of the products is 30 min. All purified products were yellow solids and had melting points of >300 °C.

Sodium 8-(Phenylamino)naphthalene-1-sulfonate (3a)

(83 mg, 63% yield) 1H NMR (400 MHz, D2O) δ 8.22 (d, J = 7.3 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.40 (d, J = 7.6 Hz, 1H), 7.33 (q, J = 7.8 Hz, 2H), 7.21 (dt, J = 12.5, 7.6 Hz, 3H), 7.01 (d, J = 7.9 Hz, 2H), 6.82 (t, J = 7.4 Hz, 1H). 13C NMR (100 MHz, D2O) δ 145.05, 137.79, 137.37, 136.82, 133.96, 129.43, 128.27, 126.55, 124.15, 123.36, 122.53, 119.90, 119.84, 116.33. HRMS (ESI) m/z calcd for C16H12NO3S– [M – H]− 298.0543; found, 298.0558.

Sodium 8-((4-Fluorophenyl)amino)naphthalene-1-sulfonate (3b)

(90 mg, 64% yield) 1H NMR (400 MHz, D2O) δ 8.29 (d, J = 7.2 Hz, 1H), 8.05 (d, J = 8.1 Hz, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.56–7.44 (m, 3H), 7.07 (dd, J = 8.2, 5.2 Hz, 2H), 7.02 (t, J = 8.7 Hz, 2H). 13C NMR (100 MHz, D2O) δ 158.24, 141.19, 138.68, 137.38, 136.87, 134.01, 128.27, 126.71, 124.25, 123.05, 122.19, 119.13, 118.34, 118.26, 115.79, 115.60, 115.56. 19F NMR (376 MHz, D2O) δ −124.35. HRMS (ESI) m/z calcd for C16H11FNO3S– [M – H]− 316.0449; found, 316.0440.

Sodium 8-((2-Fluorophenyl)amino)naphthalene-1-sulfonate (3c)

(74 mg, 53% yield) 1H NMR (400 MHz, D2O) δ 8.27 (d, J = 7.4 Hz, 1H), 8.07–8.01 (m, 1H), 7.67 (d, J = 7.6 Hz, 1H), 7.56–7.43 (m, 3H), 7.13 (t, J = 9.3 Hz, 2H), 6.95 (t, J = 7.8 Hz, 1H), 6.82 (q, J = 7.9, 7.1 Hz, 1H). 13C NMR (100 MHz, D2O) δ 154.36, 151.97, 137.13, 136.76, 136.73, 133.98, 133.56, 133.46, 128.35, 126.45, 124.32, 124.28, 124.19, 124.12, 122.84, 120.79, 119.95, 119.88, 116.11, 116.08, 115.52, 115.33. 19F NMR (376 MHz, D2O) δ −132.25. HRMS (ESI) m/z calcd for C16H11FNO3S– [M – H]− 316.0449; found, 316.0444.

Sodium 8-((3-Fluorophenyl)amino)naphthalene-1-sulfonate (3d)

(66 mg, 47% yield) 1H NMR (400 MHz, D2O) δ 8.24 (d, J = 7.4 Hz, 1H), 7.95 (d, J = 8.2 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 7.5 Hz, 1H), 7.41 (t, J = 7.9 Hz, 2H), 7.16 (q, J = 8.1 Hz, 1H), 6.79–6.69 (m, 2H), 6.52 (t, J = 8.5 Hz, 1H). 13C NMR (100 MHz, D2O) δ 164.84, 162.45, 147.50, 147.39, 137.20, 136.81, 136.71, 134.03, 130.61, 130.51, 128.55, 126.61, 124.53, 124.33, 123.04, 121.92, 111.48, 111.46, 105.74, 105.53, 101.85, 101.60. 19F NMR (376 MHz, D2O) δ −113.33. HRMS (ESI) m/z calcd for C16H11FNO3S– [M – H]− 316.0449; found, 316.0497.

Sodium 8-((3-Chlorophenyl)amino)naphthalene-1-sulfonate (3e)

(66 mg, 45% yield) 1H NMR (400 MHz, D2O) δ 8.20 (d, J = 7.3 Hz, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.38–7.25 (m, 3H), 7.15 (t, J = 7.7 Hz, 1H), 7.03 (t, J = 8.0 Hz, 1H), 6.92 (s, 1H), 6.79 (d, J = 7.9 Hz, 1H), 6.69 (d, J = 7.7 Hz, 1H). 13C NMR (100 MHz, D2O) δ 146.73, 137.28, 136.71, 136.68, 134.21, 133.89, 130.44, 128.39, 126.40, 124.26, 124.17, 122.98, 121.25, 118.85, 114.66, 114.05. HRMS (ESI) m/z calcd for C16H11ClNO3S– [M – H]− 332.0154; found, 332.0132.

Sodium 8-((4-Chlorophenyl)amino)naphthalene-1-sulfonate (3f)

(91 mg, 62% yield) 1H NMR (400 MHz, DMSO-d6) δ 10.74 (s, 1H), 8.24 (d, J = 7.3 Hz, 1H), 7.90 (d, J = 6.9 Hz, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.47 (d, J = 6.1 Hz, 1H), 7.41 (td, J = 7.7, 4.1 Hz, 2H), 7.20 (d, J = 8.8 Hz, 2H), 7.04 (d, J = 8.9 Hz, 2H).13C NMR (100 MHz, D2O) δ 143.88, 137.34, 137.29, 136.77, 133.94, 128.93, 128.35, 126.50, 124.20, 123.76, 123.22, 122.68, 120.39, 117.08. HRMS (ESI) m/z calcd for C16H11ClNO3S– [M – H]− 332.0154; found, 332.0130.

Sodium 8-((3,4-Dichlorophenyl)amino)naphthalene-1-sulfonate (3g)

(69 mg, 43% yield) 1H NMR (400 MHz, D2O) δ 8.16 (d, J = 7.4 Hz, 1H), 7.71 (d, J = 8.2 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.26 (dd, J = 14.8, 7.3 Hz, 2H), 7.12 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 8.8 Hz, 1H), 6.95 (d, J = 2.5 Hz, 1H), 6.67 (dd, J = 8.8, 2.5 Hz, 1H). 13C NMR (100 MHz, D2O) δ 145.29, 137.26, 136.68, 136.39, 133.86, 131.82, 130.46, 128.43, 126.35, 124.53, 124.19, 123.08, 121.47, 120.32, 115.90, 115.23. HRMS (ESI) m/z calcd for C16H11Cl2NO3S– [M – H]− 365.9764; found, 365.9719.

Sodium 8-((4-Bromophenyl)amino)naphthalene-1-sulfonate (3h)

(84 mg, 51% yield) 1H NMR (400 MHz, D2O) δ 8.27 (d, J = 7.4 Hz, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 7.9 Hz, 1H), 7.60 (d, J = 7.7 Hz, 1H), 7.54–7.45 (m, 2H), 7.33 (d, J = 8.6 Hz, 2H), 6.96 (d, J = 8.5 Hz, 2H). 13C NMR (100 MHz, D2O) δ 144.60, 137.21, 137.13, 136.86, 134.06, 131.91, 128.52, 126.67, 124.36, 124.18, 122.85, 121.23, 117.42, 110.48. HRMS (ESI) m/z calcd for C16H11BrNO3S– [M – H]− 375.9649; found, 375.9625.

Sodium 8-(p-Tolylamino)naphthalene-1-sulfonate (3i)

(95 mg, 69% yield) 1H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 8.21 (d, J = 7.3 Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H), 7.36 (dt, J = 12.3, 6.1 Hz, 4H), 7.08–6.96 (m, 4H), 2.24 (s, 3H). 13C NMR (100 MHz, D2O) δ 141.86, 138.92, 137.54, 136.76, 133.82, 129.84, 129.70, 127.92, 126.41, 123.91, 122.16, 121.91, 117.53, 117.45, 19.65. HRMS (ESI) m/z calcd for C17H14NO3S– [M – H]− 312.0700; found, 312.0689.

Sodium 8-((4-Methoxyphenyl)amino)naphthalene-1-sulfonate (3j)

(96 mg, 74% yield) 1H NMR (400 MHz, D2O) δ 8.25 (d, J = 8.6 Hz, 1H), 8.00 (d, J = 8.2 Hz, 1H), 7.53 (dd, J = 6.4, 2.9 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.42 (dd, J = 5.9, 2.5 Hz, 2H), 7.07 (d, J = 9.0 Hz, 2H), 6.90 (d, J = 9.0 Hz, 2H), 3.75 (s, 3H). 13C NMR (100 MHz, D2O) δ 152.97, 139.82, 138.15, 137.61, 136.77, 133.80, 127.82, 126.49, 123.95, 121.60, 121.44, 119.65, 116.37, 114.75, 55.54. HRMS (ESI) m/z calcd for C17H14NO4S– [M – H]− 328.0649; found, 328.0649.

Sodium 8-((4-Oxidophenyl)amino)naphthalene-1-sulfonate (3k)

(29 mg, 21% yield) 1H NMR (400 MHz, D2O) δ 8.26 (d, J = 6.3 Hz, 1H), 8.00 (d, J = 6.4 Hz, 1H), 7.48 (d, J = 21.7 Hz, 2H), 7.44–7.35 (m, 2H), 7.04 (d, J = 6.6 Hz, 2H), 6.87–6.78 (m, 2H). 13C NMR (100 MHz, D2O) δ 149.87, 140.07, 137.86, 137.41, 136.93, 134.05, 128.15, 126.89, 124.28, 121.98, 121.42, 120.40, 117.29, 116.20. HRMS (ESI) m/z calcd for C16H12NO4S– [M – H]− 314.0493; found, 314.0520.

Sodium 8-((4-Cyanophenyl)amino)naphthalene-1-sulfonate (3l)

(50 mg, 35% yield) 1H NMR (400 MHz, D2O) δ 8.19 (d, J = 7.4 Hz, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.27–7.20 (m, 3H), 7.14 (t, J = 7.8 Hz, 1H), 6.74 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz, D2O) δ 149.71, 137.24, 136.57, 134.79, 133.82, 133.80, 128.74, 126.10, 125.76, 124.41, 123.59, 123.16, 121.52, 113.88, 98.04. HRMS (ESI) m/z calcd for C17H11N2O3S– [M – H]− 323.0496; found, 323.0417.

Sodium 8-((4-Nitrophenyl)amino)naphthalene-1-sulfonate (3m)

(24 mg, 16% yield) 1H NMR (400 MHz, D2O) δ 8.23 (d, J = 7.2 Hz, 1H), 7.98–7.83 (m, 3H), 7.70–7.59 (m, 1H), 7.49–7.30 (m, 3H), 6.77 (d, J = 8.5 Hz, 2H). 13C NMR (100 MHz, D2O) δ 152.51, 137.51, 137.02, 136.54, 133.99, 133.93, 128.99, 126.88, 126.61, 126.30, 124.64, 124.57, 123.80, 112.90. HRMS (ESI) m/z calcd for C16H11N2O5S– [M – H]− 343.0394; found, 343.0365.

Sodium 8-([1,1′-Biphenyl]-4-ylamino)naphthalene-1-sulfonate (3n)

(38 mg, 23% yield) 1H NMR (400 MHz, DMSO-d6) δ 10.81 (s, 1H), 8.24 (d, J = 6.1 Hz, 1H), 7.89 (d, J = 7.1 Hz, 1H), 7.62 (d, J = 7.3 Hz, 2H), 7.57–7.47 (m, 4H), 7.41 (q, J = 8.0 Hz, 4H), 7.27 (t, J = 7.4 Hz, 1H), 7.15 (d, J = 8.6 Hz, 2H). 13C NMR (100 MHz, D2O) δ 144.86, 140.36, 137.43, 137.29, 136.92, 134.10, 131.63, 129.09, 128.49, 127.69, 126.74, 126.10, 124.36, 123.86, 120.78, 116.39. HRMS (ESI) m/z calcd for C22H16NO3S– [M – H]− 374.0856; found, 374.0866.

Sodium 8-((4-Acetamidophenyl)amino)naphthalene-1-sulfonate (3o)

(94 mg, 60% yield) 1H NMR (400 MHz, D2O) δ 8.26 (d, J = 6.4 Hz, 1H), 8.03 (d, J = 8.3 Hz, 1H), 7.63 (d, J = 8.5 Hz, 1H), 7.58 (d, J = 7.7 Hz, 1H), 7.47 (q, J = 7.7 Hz, 2H), 7.18 (d, J = 8.8 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 2.07 (s, 3H). 13C NMR (100 MHz, D2O) δ 172.92, 143.07, 137.83, 137.31, 136.95, 134.11, 128.84, 128.48, 126.79, 124.39, 124.09, 123.70, 122.58, 120.47, 116.56, 22.49. HRMS (ESI) m/z calcd for RP-C18H15N2O4S– [M – H]− 355.0758; found, 355.0750.

Fluorescence Testing Procedures

Using a BMG ClarioStar multimode fluorescence, luminescence, and absorbance plate reader and Corning 3882 plates, the fluorescence properties of ANS and its derivatives were evaluated. Each compound was evaluated in 100 μL volume at 100 μM concentration in both water and ethylene glycol. For each compound, a full absorbance spectrum was measured after blanking with the appropriate solvent from 310 to 600 nm. Likewise, maximum emission wavelengths were measured from 400 to 650 nm, except for 3m, which was observed from 450 to 650 nm due to a high maximum absorption wavelength. The centers of the most major peaks were taken as the maximum wavelengths for both absorption and emission; a few compounds had minor peaks in the ranges detected. Fluorescence emission was measured by exciting at an optimized gain of 2098 at the maximum absorption wavelength. Because of instrument limitations, some compounds with low wavelength absorption wavelengths were excited at 320 nm. In addition, some of these same compounds had overlapping peaks from the absorption in the UV region, making it difficult to precisely determine the absorption maxima in these cases. These changes are noted in Table . Compounds with high fluorescence yields in ethylene glycol, particularly 3b, 3c, 3d, 3e, 3g, and 3l, had to be excited with a gain of 1049 so their emission spectra could be recorded within the instrument parameters.

As a positive control, the properties of ANS (3a) were evaluated. The maximum absorbance wavelengths of ANS matched well with what was previously reported in both water and ethylene glycol.[16] The maximum emission wavelength of ANS matches well in ethylene glycol and is in between two reported values for the maximum emission wavelength in water.[16,26] Fluorescence yields were normalized to previously reported values of ANS fluorescence yields, which matched well with what had been observed in water and ethylene glycol.[16] For each gain used on the instrument, a conversion constant was calculated to correlate the amplitudes observed in the absorption spectra to the amplitudes observed in the emission spectra. An enhancement factor for each compound was calculated as a ratio of the fluorescence yield in ethylene glycol to the fluorescence yield in water.