Simultaneous Determination of Concentration and Enantiomeric

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Simultaneous Determination of Concentration and Enantiomeric Composition in Fluorescent Sensing Lin Pu* Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States CONSPECTUS: Fluorescent sensors have found broad applications in determining the concentrations of diverse analytes by measuring specific sensor−analyte fluorescent responses. For a chiral substrate containing varying composition of two enantiomers, both the concentration and enantiomeric composition should greatly influence the fluorescent response of an enantioselective fluorescent sensor. Thus, multiple independent measurements are normally needed to determine both the concentration and enantiomeric composition of a chiral compound. In order to facilitate the application of the enantioselective fluorescent sensors, our laboratory has developed four strategies to simultaneously determine the concentration and enantiomeric composition of various chiral substrates by a single fluorescence measurement. A mixture of a chiral BINOL-based dialdehyde and an achiral compound salicylaldehyde in the presence of Zn2+ is used to interact with chiral diamines, amino alcohols, and amino acids. The fluorescence enhancement at λ1 = 447 nm due to the achiral sensor is mostly determined by the concentration of the substrates, and the fluorescence enhancement at λ2 > 500 nm due to the chiral sensor is highly enantioselective. A 3D graph combining the fluorescence intensities at λ1 and λ2 can be used to determine the enantiomeric composition. A chiral conjugated polymer containing the BINOL−dialdehyde units is shown to amplify the enantioselectivity of the small molecule sensor under the same conditions. Combination of the chiral polymer with salicylaldehyde allows simultaneous concentration and enantiomeric composition determination. In a pseudoenantiomeric sensor pair of the BINOL-based amino alcohols, one sensor shows greater fluorescence enhancement with one enantiomer of chiral α-hydroxy carboxylic acid at λ1 = 374 nm and another sensor shows greater fluorescence enhancement with another enantiomer at λ2 = 330 nm. Using a mixture of this sensor pair allows the determination of both concentration and enantiomeric composition with one fluorescence measurement. A BINOL-based trifluoromethyl ketone is found to exhibit dual emission responses toward a chiral diamine at λ1 = 370 nm and λ2 = 438 nm. The fluorescence enhancement at λ1 is mostly determined by the substrate concentration and that at λ2 is highly enantioselective. Thus, using one sensor with one measurement gives both parameters. A BINOL−naphthyl imine compound is designed to show two different fluorescent responses toward functional chiral amines in the presence of Zn2+. When the naphthylamine unit is displaced off the sensor by a chiral amine substrate via imine metathesis, the emission of naphthylamine is restored at λ1 = 427 nm, which allows determination of the substrate concentration. The fluorescence enhancement at λ2 > 500 nm due to the formation of the new chiral imine products is highly enantioselective. The work discussed here has provided convenient methods to obtain the two important parameters of a chiral molecule by a single fluorescence measurement. They should contribute to the development of analytical tools for the rapid assay of chiral compounds.

1. INTRODUCTION With the extensive applications of fluorescence molecular sensors in analytical chemistry,1,2 there is also growing research on enantioselective fluorescence discrimination of chiral organic compounds3−6 because of the importance of chiral molecules in organic synthesis, pharmaceutical compounds, materials components, and agricultural products. In the past decade, significant progress has been made in the development of enantioselective fluorescence sensors for potential applications in the rapid analysis of chiral organic compounds. These molecular sensors are generally constructed by incorporating one or more fluorescence response mechanisms into a chiral © 2017 American Chemical Society

molecular receptor to sense the corresponding substrates. The intermolecular interactions including hydrogen bonding, π−π interaction, electrostatic attraction, metal coordination, and covalent bond formation between a receptor and a chiral substrate can generate a variety of fluorescence responses such as quenching, enhancement, shift of the emission wavelength, changing of the excited state lifetime, excimer emission, etc. The different responses of the fluorescence sensor toward the Received: January 18, 2017 Published: March 13, 2017 1032

DOI: 10.1021/acs.accounts.7b00036 Acc. Chem. Res. 2017, 50, 1032−1040

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Accounts of Chemical Research

wavelength (λ2 > 500 nm). Both (R)-1 and SA are almost nonfluorescent with or without Zn2+. When they are treated with a functional amine such as phenylalaninol in the presence of Zn2+, an imine−Zn(II) complex like 2 can be generated to give large fluorescence enhancement due to a restricted excited state isomerization of the imine bond and a disrupted excited state proton transfer. When the chiral sensor is used, the chirality matched sensor−substrate reaction leads to greater fluorescence enhancement than the mismatched one.

two enantiomers of the chiral substrate lead to their discrimination. Since both the concentration and the enantiomeric composition of a chiral substrate can strongly influence the fluorescence response of an enantioselective fluorescence sensor, multiple independent measurements are required in order to determine these two parameters. This will not be convenient when the enantioselective fluorescence sensors are applied to the high throughput assay of chiral molecules. For example, Wolf reported the use of the combination of racemic and enantiomerically pure chiral sensors to carry out tandem UV and fluorescence measurements, CD and UV measurements, or CD and fluorescence measurements.7−9 In order to reduce the two optical measurements to one, Anslyn used a dual-chamber quartz cuvette to simultaneously measure the UV responses of an indicator-displacement sensor toward chiral substrates in two samples in which one sample contains a chiral sensor and another sample contains an achiral sensor with responses at two different wavelengths.10,11 Since early 2000, our laboratory has been systematically developing fluorescence sensors for the enantioselective recognition of chiral organic molecules.12−20 Using 1,1′-bi-2naphthol (BINOL) as the chirality source as well as the core fluorophore, we have constructed structurally diverse chiral fluorescence sensors. Highly enantioselective fluorescence sensors have been discovered for the recognition of chiral substrates such as α-hydroxycarboxylic acids, amines, diamines, amino alcohols, and amino acids. Recently, in order to facilitate the application of these sensors in fluorescence assay and simplify the experimental operation, we have developed several strategies to simultaneously quantify the concentration and enantiomeric composition of chiral molecules by one fluorescence measurement. Herein, these strategies are discussed.

A 1:4 mixture of (R)-1 and SA in methanol in the presence of Zn(OAc)2 (2 equiv) was used to interact with L- and Dphenylalaninol. As shown in Figure 1a, L-phenylalaninol

Figure 1. Fluorescence spectra of (R)-1 + SA (1:4, total concentration 5.0 × 10−5 M in methanol/1% CH2Cl2) + Zn2+ (1.0 × 10−4 M) with Lphenylalaninol (a) and D-phenylalaninol (b) [(0−6.0) × 10−5 M]. Reference 22. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.

2. USING A CHIRAL AND AN ACHIRAL SENSOR PAIR

generated large fluorescence enhancements at both λ1 and λ2, but D-phenylalaninol only led to major fluorescence enhancement at the short wavelength λ1 as shown in Figure 1b. This chiral and achiral sensor pair was treated with phenylalaninol at varying concentrations and enantiomeric compositions. Figure 2a shows that the fluorescence intensity of the sensor at λ1 is only slightly influenced by the enantiomeric composition and is mostly determined by the total concentration of the substrate. Figure 2b shows that the fluorescence intensity at λ2 is strongly influenced by both the concentration and enantiomeric composition of the amino alcohol substrate. A 3D graph is obtained by plotting the fluorescence intensities at λ1 and λ2 against the composition of L-phenylalaninol in the enantiomeric mixture to give Figure 2c. Using this plot, the enantiomeric composition of the amino alcohol can be determined by one fluorescence measurement. The fluorescence intensities at λ1 and λ2 were also plotted against the total concentration of the D- and L-phenylalaninol to obtain the 3D graph Figure 2d. This graph takes into consideration the small effect of the enantiomeric composition on the fluorescence intensity at λ1, and it allows the determination of the total concentration of the substrate. Therefore, both the concentration and the enantiomeric composition of the chiral amino alcohol can be determined by one fluorescence measurement. The (R)-1 + SA + Zn2+ fluorescence sensor system can be applied to simultaneously determine the concentration and enantiomeric composition of a variety of functional chiral amines including amino alcohols, diamines, and amino acids.

2.1. A Small Molecule-Based Sensor

The BINOL-based chiral aldehyde (R)-1 shows little fluorescence in methanol. This is attributed to an efficient excited state proton transfer between the hydroxyl groups and the adjacent carbonyls, which quenches the fluorescence of BINOL. We found that the methanol solution of (R)-1 in the presence of Zn2+ shows highly enantioselective fluorescence enhancement at λ > 500 nm when treated with functional chiral amines including diamines, amino alcohols, and amino acids.21

In order to simultaneously determine the concentration and enantiomeric composition of functional chiral amines, we studied the use of the (R)-1-based chiral sensor in combination with an achiral sensor to conduct the fluorescence assay.22 We found that salicylaldehyde (SA), an achiral molecule, in combination with Zn2+ shows greatly enhanced fluorescence at λ1 = 447 nm in the presence of both D- and L-phenylalaninol. That is, SA can be used as a fluorescent sensor to determine the concentration of phenylalaninol and other functional amines at a shorter wavelength, and (R)-1 can be used to determine the enantiomeric composition of these substrates at a longer 1033


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quenching sites have reacted. If there is one unreacted binding site on the polymer chain, the fluorescence of the polymer can still be quenched because of the energy migration along the conjugated polymer chain. That is, the fluorescence enhancement of this type of conjugated polymer requires a cumulative effect of the substrates on the binding sites. This effect should enhance the enantioselectivity of the polymer over each binding site. That is, if a small molecule-based sensor can be incorporated into a conjugated polymer as the fluorescence quenching binding sites, its enantioselectivity in fluorescence sensing can be greatly increased due to the energy migration of the conjugated polymer. For an enantioselective fluorescence response of IR/IS (>1) at each binding site toward the (R)- and (S)-enantiomer of a chiral substrate, the polymer might exhibit a greatly enhanced enantioselectivity of (IR/IS)n (n = number of the binding sites on the conjugated polymer chain). For example, if the enantioselective fluorescence response at each binding site could be correlated with the equilibrium constant ratio KR/KS (≠ 1), the overall enantioselective fluorescence response of the polymer could be correlated with (KR/KS)n. Thus, a small enantioselectivity at one binding site can be greatly amplified in a conjugated polymer structure due to the cumulative effect. We have synthesized the BINOL-based main chain chiral conjugated polymer (S)-6 by conducting a Suzuki coupling of a BINOL-based monomer (S)-5 with an aryldiboronic acid (Scheme 1). The molecular weight of this polymer is Mn =

Figure 2. Fluorescence response of (R)-1 + SA (1:4, total concentration 5.0 × 10−5 M in methanol/1% CH2Cl2) + Zn2+ (1.0 × 10−4 M) versus the enantiomeric purity of phenylalaninol at various concentrations at λ1 (a) and λ2 (b) and the 3D plots (c, d). Reference 22. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.

2.2. A Chiral Conjugated Polymer-Based Fluorescence Sensor23

The strategy to use a chiral and achiral sensor pair is also applied to a chiral polymer-based sensor. Using polymers in fluorescence sensing has a number of potential advantages. For example, the polymer-based sensors can be used to selectively react with the products generated from the catalyst screening reactions, which can then be separated from the reaction medium by filtration, precipitation/filtration, or membrane filtration. This should minimize the interference of the reagents, catalysts, and other components in the reaction mixture with the subsequent optical measurement. Polymers can be also used to make film- or membrane-supported sensors. We recently proposed to use the main chain chiral conjugated polymers to enhance the enantioselectivity of fluorescence sensors in chiral recognition. As shown in Figure 3, the chiral conjugated polymer 3 is nonfluorescent because of its fluorescence quenching binding sites. When a chiral substrate reacts with the binding sites of polymer 3 to inhibit the fluorescence quenching, the fluorescence of the resulting polymer 4 can be turned on only after all the fluorescence

Scheme 1. Synthesis of the Main Chain Chiral Conjugated Polymer (S)-6

89 000 (PDI = 1.35), and it is soluble in methylene chloride, chloroform, and THF but insoluble in methanol and water. In methylene chloride solution, (S)-6 is almost nonfluorescent. The monomeric compound (R)-1 in combination with Zn2+ shows highly enantioselective fluorescence responses toward a variety of functional chiral amines in methanol solution, but its enantioselectivity in methylene chloride solution is diminished. We have examined the use of polymer (S)-6 in combination with Zn2+ for the fluorescent recognition of chiral amino alcohols in methylene chloride. This polymer-based sensor has exhibited greatly enhanced enantioselectivity over the small molecule under the same conditions. That is, the cumulative effect of the polymer amplifies the small selectivity at each binding site. It demonstrates that the energy migration in a conjugated polymer can be used to enhance the selectivity of a small molecule-based sensor.

Figure 3. Turn on fluorescence of a chiral conjugated polymer. 1034


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Accounts of Chemical Research We studied the use of (S)-6 in combination with the achiral sensor SA to simultaneously determine the concentration and enantiomeric composition of amino alcohols. A 1:2 mixture of (S)-6 and SA in CH2Cl2 was prepared and combined with 2 equiv of Zn(OAc)2. This mixture had only very weak fluorescence. When the chiral polymer and achiral molecule sensor mixture interacted with the amino alcohol (R)- and (S)leucinol, large fluorescence enhancements at two emission wavelengths of λ1 ≈ 430 nm and λ2 ≈ 530 nm were observed (Figure 4a,b). The fluorescence enhancement at λ1 is due to the

Figure 5. Fluorescence response of (S)-6 + SA (1:2, total concentration 1.5 × 10−4 M in CH2Cl2) + Zn2+ (3.0 × 10−4 M) versus the enantiomeric purity of leucinol at various concentrations at λ1 (a) and λ2 (b) and the 3D plots (c, d). Reproduced from ref 23 with permission from the Royal Society of Chemistry.

good enantioselective fluorescence response toward mandelic acid (MA). Figure 6 shows that when (S)-7 is treated with (R)-

Figure 4. Fluorescence response of (S)-6 + SA (1:2, total concentration 1.5 × 10−4 M in CH2Cl2) + Zn2+ (3.0 × 10−4 M) toward (R)- and (S)-leucinol. Reproduced from ref 23 with permission from the Royal Society of Chemistry.

reaction of SA with the amino alcohol and Zn2+ to form a salicylimine−Zn2+ complexes, and that at λ2 is due to the reaction of (S)-6 to form similar complexes of more extended conjugation. Figure 4c shows that the fluorescence intensity at λ1 is mostly determined by the concentration of the amino alcohol with only slight influence by the chiral configuration. Figure 4d shows good enantioselective fluorescence response at λ2. The fluorescence responses of the (S)-6 + SA + Zn2+ sensor system toward leucinol at varying enantiomeric compositions at selected concentrations were studied. Figure 5a,b plots (R)leucinol percentage at various concentration against the fluorescence intensity at λ1 and λ2, respectively. On the basis of these plots, two 3D graphs of the fluorescence intensities at λ1 and λ2 versus the concentration and (R)-leucinol percentage, respectively, were obtained as shown in Figure 5c,d. Using Figure 5c,d allows determination of both concentration and enantiomeric composition with one fluorescence measurement.

Figure 6. (a) Fluorescence spectra of (S)-7 (1.0 × 10−4 M, CH2Cl2) with or without MA (4.0 × 10−3 M). (b) Fluorescence enhancement of (S)-7 (1.0 × 10−4 M, CH2Cl2) at λ1 = 374 nm versus [MA]. Reproduced from ref 19. Copyright 2010 American Chemical Society.

MA, there is a large fluorescence enhancement at λ1 = 374 nm, but much smaller fluorescence enhancement is observed in the presence of (S)-MA. Figure 7 shows that when (R)-8 is treated with (S)-MA, there is a large fluorescence enhancement at a shorter wavelength of λ2 = 330 nm because of the reduced conjugation of H8BINOL, but much smaller fluorescence enhancement is observed in the presence of (R)-MA. The fluorescence enhancement generated by the chirality matched sensor−substrate interaction is attributed to two major factors: (a) Protonation of the amine nitrogens of the sensors by the substrates disrupts their intramolecular hydrogen bonding with the aryl hydroxyl groups and inhibits the fluorescence quenching generated by the excited state proton transfer. (b) The chirality matched sensor−substrate interaction can form a structurally more rigid intermolecular complex, leading to greater fluorescence enhancement. Because of the distinctively different responding fluorescent wavelengths between (S)-7 and (R)-8, a 1:1 mixture of this pseudoenantiomeric pair in CH2Cl2 (each at 1.0 × 10−4 M) was

3. USING A PSEUDOENANTIOMERIC SENSOR PAIR19 Compounds (S)-7 and (R)-8 are a pseudoenantiomeric sensor pair with the opposite chiral configurations at the axially chiral biaryl unit and the chiral amine carbons, and they have very different fluorescence response wavelengths. Compound (S)-7 is derived from BINOL, and (R)-8 is derived from the partially hydrogenated BINOL, H8BINOL. Both (S)-7 and (R)-8 exhibit 1035


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is nonfluorescent in CH2Cl2. When it was treated with the two enantiomers of trans-1,2-cyclohexanediamine, (R,R)- and (S,S)10, large fluorescence enhancements at λ1 = 370 nm and λ2 = 438 nm were observed. As shown in Figure 9, both (R,R)- and

Figure 7. (a) Fluorescence spectra of (R)-8 (1.0 × 10−4 M, CH2Cl2) with or without (R)- and (S)-MA (4.0 × 10−3 M). (b) Fluorescence enhancement of (R)-8 (1.0 × 10−4 M, CH2Cl2) at λ2 = 330 nm versus [MA]. Reproduced from ref 19. Copyright 2010 American Chemical Society.

Figure 9. Fluorescence spectra of (S)-9 (1.0 × 10−5 M) with or without (R,R)- and (S,S)-10 (5.0 × 10−3 M) (Solvent CH2Cl2). Reproduced from ref 24a. Copyright 2012 American Chemical Society.

(S,S)-10 caused similar fluorescence enhancement for (S)-9 at λ1, but (R,R)-10 increased the fluorescence intensity at λ2 much greater than (S,S)-10. That is, at λ1, the fluorescence response of (S)-9 is not sensitive to the chiral configuration of the diamine, which can be used to determine the concentration of the substrate, and at λ2, the fluorescence response is highly enantioselective, which can be used to determine the enantiomeric composition. We have plotted the fluorescence intensity ratio I1/I2 at the two emission wavelengths of (S)-9 versus the concentration of (R,R)- and (S,S)-10. As shown in Figure 10, this ratio is found

used to interact with MA of varying concentrations and enantiomeric compositions. The fluorescence intensity difference at λ1 (374 nm) and λ2 (330 nm), that is (I1/I10 − I2/I20) (I1 and I2 indicate fluorescence intensity at λ1 and λ2 in the presence of the acid, and I10 and I20 indicate fluorescence intensity in the absence of the acid) and the fluorescence intensity sum (I1/I10 + I2/I20) are plotted against the acid concentration (Figure 8a) and (R)-acid percentage (Figure 8b),

Figure 10. Plot of I1/I2 for (S)-9 (1.0 × 10−5 M) in the presence of varying concentrations of (R,R)- and (S,S)-10 (Fluorescence intensity I1 at λ1 = 370 nm and I2 at λ2 = 438 nm. solvent, CH2Cl2). Reproduced from ref 24a. Copyright 2012 American Chemical Society. Figure 8. 3D plots of (I1/I10 − I2/I20) and (I1/I10 + I2/I20) versus MA concentration (mM) (a) and (I1/I10 − I2/I20) and (I1/I10 + I2/I20) versus (R)-acid percentage (b). Reproduced from ref 19. Copyright 2010 American Chemical Society.

to be independent of the concentration of the substrate, and each enantiomer of the substrate corresponds to a constant I1/ I2 ratio. For (S,S)-10, the I1/I2 ratio is 2.60, and for (R,R)-10, it is 0.67. The sensor (S)-9 was interacted with the chiral diamine with varying concentrations and enantiomeric compositions. Figure 11 plots the fluorescence intensity ratio I1/I2 at various total concentrations of the two enantiomers versus (S,S)-10 percentage. It shows that the total concentration of the substrate has little influence on the fluorescence intensity ratio and using I1/I2 can directly determine the enantiomeric composition of the diamine. That is, the enantiomeric composition of the chiral compound can be determined by one fluorescence measurement without the need to know the sample concentration. We also obtained a 3D graph for the concentration of the chiral diamine versus the fluorescence intensity λ1 and the fluorescence intensity ratio I1/I2 (Figure 12). The incorporation of I1/I2 into the plot is to correct the small effect of the chiral

respectively. Using these 3D graphs allows the determination of both the concentration and enantiomeric composition of a MA sample by a single fluorescence measurement.

4. DISCOVERY OF USING ONE FLUORESCENCE SENSOR TO DETERMINE TWO PARAMETERS24 The above two strategies use a mixture of two fluorescence sensors, each of which has a different emission wavelength, to determine the concentration and enantiomeric composition of chiral substrates. We have also discovered that it is possible to use one fluorescence sensor to simultaneously determine the two parameters. Compound (S)-9 containing two trifluoroacetyl groups at the 3,3′-positions of BINOL was synthesized. This compound 1036


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chiral compound by one fluorescence measurement. We have taken on the challenge to develop a general strategy to rationally design such sensors. Scheme 2 shows our proposed strategy to construct a sensor with dual emission wavelengths at which they respond Scheme 2. Proposed General Strategy To Convert an Enantioselective Fluorescence Sensor to a Dual Responsive Sensor

Figure 11. Plot of I1/I2 vs (S,S)-10 percentage at various diamine concentrations (solvent, CH2Cl2). Reproduced from ref 24a. Copyright 2012 American Chemical Society.

differently toward the enantiomers of a chiral substrate. An achiral fluorophore A with emission at λ1 will be linked with an enantioselective fluorescence sensor that shows fluorescence response at λ2 (λ1 ≠ λ2). A fluorescence quenching mechanism will be incorporated into the linker to make the resulting probe [A + B] nonemissive at both λ1 and λ2. When the adduct [A + B] is treated with a chiral substrate, which can cleave the linker and release A, the fluorescence at λ1 should be turned on. If this linker cleavage is not enantioselective, the emission at λ1 should be only dependent on the concentration of the substrate not its enantiomeric composition. The cleavage of the linker in [A + B] should also restore the enantioselective fluorescence response of B toward the chiral substrate at λ2. Thus, it is possible to use the probe [A + B] to simultaneously determine both concentration and enantiomeric composition with one fluorescence measurement. On the basis of the above proposed strategy, compound (R)12 was synthesized from the condensation of (R)-1 with 2naphthylamine (Scheme 3), since 2-naphthylamine shows

Figure 12. Plot of I1 and I1/I2 vs the total concentration of 10 with various enantiomeric compositions. Reproduced from ref 24a. Copyright 2012 American Chemical Society.

configuration on the fluorescence intensity at λ1. As shown in Figure 12, the concentration of the substrate can be determined by measuring the fluorescent intensity at λ1 and λ2. This work demonstrates that using one fluorescence sensor can simultaneously determine both the concentration of a chiral molecule and the enantiomeric composition by a single fluorescence measurement. A sensor−substrate adduct like 11 is proposed to explain the observed dual emissions of (S)-9 when treated with the chiral diamine. Addition of an amine group at one of the carbonyl carbons forms a hemiaminal, which breaks the conjugation between the carbonyl group and the aromatic ring and also disrupts the original hydrogen bonding between the BINOL hydroxyl group and the carbonyl oxygen. This should turn on the fluorescence of the naphthol unit at the short wavelength λ1. The observed little enantioselectivity at λ1 can be attributed to the location of the carbonyl group, which is far away from the chiral core. In 11, the second amine group of the diamine can interact with the acidic aryl OH groups at the BINOL core. The base-promoted polarization or deprotonation of the BINOL unit is expected to generate the long wavelength emission at λ2. The proximity between the chiral core of the BINOL unit and the chiral amino carbon center in this second interaction can contribute to the observed high enantioselectivity at the long wavelength emission.

Scheme 3. Preparation of (R)-12

emission at λ1 = 427 nm and (R)-1 in combination with Zn2+ shows enantioselective fluorescent response to functional chiral amines at λ2 > 500 nm. Because of the intramolecular hydrogen bonding between the BINOL hydroxyl groups and the imine nitrogens in (R)-12, the fluorescence of 2naphthylamine is quenched. In the presence of Zn2+, (R)-12 shows only weak emission at 555 nm (Figure 13). When the (R)-12 + Zn2+ solution was treated with the chiral diamine (R,R)- and (S,S)-10, large fluorescence enhancement was observed at λ1 and λ2 as shown in Figure 13. At λ1 = 427 nm, both enantiomers of the diamine gave almost the same emission indicating nonenantioselective displacement of 2-naphthylamine off (R)-12 to restore the emission of 2-naphthylamine. At λ2 > 500 nm, a highly

5. A RATIONAL DESIGN FOR USING ONE FLUORESCENCE SENSOR TO DETERMINE TWO PARAMETERS25 The sensor (S)-9 described in the above section is a unique example that can be used as a single sensor to simultaneously measure the concentration and enantiomeric composition of a 1037


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Figure 13. Fluorescence spectra of (R)-12 (2.0 × 10−5 M in CH3OH/ 2% CH2Cl2) with Zn(OAc)2 (2 equiv) (green) in the presence of (S,S)-10 (7.5 equiv) (red) or (R,R)-10 (7.5 equiv) (blue). Reproduced from ref 25. Copyright 2015 American Chemical Society.

enantioselective fluorescence response was observed with (S,S)10 giving much greater fluorescence enhancement than (R,R)10. The fluorescence response at λ2 was the same as that observed when (R)-1 was used directly to interact with the diamine in the presence of Zn2+. The reaction of (R)-12 with a chiral amine in the presence of Zn2+ is proposed in Scheme 4.

Figure 15. Fluorescence response of (R)-12 (2.0 × 10−5 M in CH3OH/2% CH2Cl2) + Zn2+ (2 equiv) toward L-phenylglycinol (a) and D-phenylglycinol (b). I427 (c) and I527 (d) versus the concentrations of L- and D-phenylglycinol. Reproduced from ref 25. Copyright 2015 American Chemical Society.

Scheme 4. Proposed Imine Metathesis for the Reaction of (R)-12 + Zn2+ with a Chiral Amine

the sensor with D-phenylglycinol also enhanced emissions at 427 and 505 nm, but the emission at 505 nm was much lower than that caused by L-phenylglycinol. The fluorescence intensity at λ1 = 427 nm is plotted against the concentrations of L- and Dphenylglycinol in Figure 15c, which indicates small enantioselectivity in the displacement of 2-naphthylamine by this amino alcohol, that is, the chiral configuration of the amino alcohol has certain effects on I427. The fluorescence intensity at 527 nm is plotted against the concentration of L- and D-phenylglycinol in Figure 15d, which shows highly enantioselective response. The sensor solution of (R)-12 + Zn2+ was interacted with phenylglycinol of varying concentrations and enantiomeric compositions. A 3D graph was obtained for the concentration of the amino alcohol versus the fluorescence intensity at λ1 = 427 nm and λ2 = 527 nm. This 3D plot takes into the consideration of the effect of the chiral configuration of the amino alcohol on the fluorescence intensity at λ1. The fluorescence intensities at λ1 and λ2 are also plotted against the L-phenylglycinol percentage in Figure 16b. Therefore, using Figures 16a,b can determine both the concentration and enantiomeric composition of the amino alcohol by one fluorescence measurement. The interaction of (R)-12 + Zn2+ with a variety of chiral amino acids was conducted in the presence of Bu4NOH (amino

Compound (R)-12 was interacted with the diamine in various concentrations and enantiomeric compositions. It was found that at diamine concentration greater than 7 equiv of (R)-12, the fluorescence intensity ratio I525/I500 is only influenced by the enantiomeric composition but independent of the concentration of the substrate as shown in Figure 14a.

Figure 14. Fluorescence responses of (R)-12 (2.0 × 10−5 M in CH3OH/2% CH2Cl2) + Zn2+ (2 equiv) toward 10: (a) I525/I500 versus (S,S)-10 percentage at various concentrations. (b) I427 versus the concentrations of 10 at various enantiomeric compositions. Reproduced from ref 25. Copyright 2015 American Chemical Society.

Thus, using I525/I500 allows direct determination of the enantiomeric composition. The fluorescence intensity at λ1 = 427 nm also allows direct determination of the concentration of the diamine (Figure 14b). The interaction of the (R)-12 + Zn2+ sensor with chiral amino alcohols such as phenylglycinol was studied. Similar to the reaction with the diamine 10, these amino alcohols caused dual emission of the sensor. As shown in Figure 15a,b, addition of L-phenylglycinol to (R)-12 + Zn2+ turned on the fluorescence at λ1 = 427 nm and λ2 = 527 nm. Treatment of

Figure 16. Fluorescence responses of (R)-12 (2.0 × 10−5 M in CH3OH/2% CH2Cl2) + Zn2+ (2 equiv) toward phenylglycinol: (a) I427 and I527 versus the total concentration of phenylglycinol. (b) I427 and I527 versus L-phenylglycinol percentage. Reproduced from ref 25. Copyright 2015 American Chemical Society. 1038


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screening reactions for the subsequent fluorescence measurement.23 (3) Enantioselective fluorescence imaging of chiral molecules in biological systems such as in living cells is under investigation aiming at probing the biological functions of the chiral compounds.27 Besides our work described in this paper, Heemstra and coworkers reported the use of the DNA-based fluorescent sensors to simultaneously determine the concentration and enantiomeric composition of chiral compounds.28 More and more researchers are also working in the field of enantioselective fluorescent sensing as represented in a few selected recent publications.29−35 With the joint efforts of all the researchers in this active research area, we see a bright future for the enantioselective fluorescent sensors.

acid/n-Bu 4NOH 1:1.1) in methanol, and similar dual fluorescence enhancements were observed. We have demonstrated that both the concentration and enantiomeric composition of the amino acids can be simultaneously determined by using this sensor system.

6. SUMMARY AND OUTLOOK In this Account, we have described four strategies to use fluorescence sensors to simultaneously determine the concentration and enantiomeric composition of chiral compounds: (1) A mixture of an achiral sensor with fluorescence response at λ1 and a chiral sensor with fluorescence response at a different wavelength λ2 was used. The achiral sensor can report the concentration of the substrates at λ1 since this response is nearly independent of the chiral configuration of the substrate. The chiral sensor shows enantioselective fluorescence response to the chiral substrate at λ2. (2) A pesudoenantiomeric sensor pair is used in which one sensor mainly reports the fluorescence response of one enantiomer at λ1 and another one mainly reports the fluorescence response of the opposite enantiomer at a distinctively different λ2. The difference between the two fluorescence intensities (I1 − I2) is correlated with the enantiomeric composition and their sum (I1 + I2) is correlated with the concentration. (3) A fluorescence sensor is discovered to respond to a chiral substrate in very different way at two emission wavelengths. The fluorescence enhancement at λ1 is correlated with the concentration of the substrate with little influence by its chiral configuration. At λ2, the fluorescence enhancement is highly enantioselective. (4) A fluorescence sensor with two distinctively different emission wavelengths is obtained by a rational design. This sensor is constructed by linking an achiral and a chiral fluorophore together to achieve a fluorescence quenched state. The achiral fluorophore can be displaced off by a chiral substrate to show nonenantioselective fluorescence enhancement at λ1, and the remaining chiral fluorophore shows enantioselective fluorescence enhancement at λ2. All four types of sensor systems share one common feature; that is, they all exhibit two distinctively different emission wavelengths each of which responds to the two enantiomers of a chiral substrate very differently. Generally, two 3D graphs can be produced to correlate the fluorescence responses at the two wavelengths with the concentration and the enantiomeric composition, respectively. Using these 3D graphs allows determination of both concentration and enantiomeric composition of a chiral substrate by one fluorescence measurement. These methods should facilitate the application of the enantioselective fluorescence sensors in high throughput chiral assay. The chiral substrates studied in this research include αhydroxy carboxylic acids, diamines, amino alcohols, and amino acids. In order to further expand the scope of the substrates, new sensors will be designed and investigated. In addition, we are also venturing in the field of enantioselective fluorescence sensing along three new directions: (1) In order to minimize the interference of the reagents, catalysts, and other components with the fluorescence measurement when an enantioselective fluorescence sensor is applied in high throughput chiral catalyst screening, we are developing enantioselective fluorescence recognition in fluorous phase.26 (2) Chiral conjugated polymer-based sensors are under investigation in order to enhance the enantioselectivity of the small molecule-based sensors and to facilitate the separation of the chiral products from the reaction medium of catalyst

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AUTHOR INFORMATION

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*E-mail: [email protected]. ORCID

Lin Pu: 0000-0001-8698-3228 Author Contributions

I thank all of my students, postdoctoral associates, and visiting scholars as well as my collaborators, whose names are cited in the references, for their great contributions to the research described in this Account. The support of our research from the US NSF and ACS-PRF is gratefully acknowledged. Notes

The author declares no competing financial interest. Biography Lin Pu was born in 1965 in Xuyong, Sichuan Province, China. He received his B.S. degree in chemistry from Sichuan University in 1984. He then obtained the Doering Fellowship (CGP) to undertake graduate study in the department of chemistry at University of California, San Diego, in 1985. Under the supervision of Professor Joseph M. O’Connor, he obtained his Ph.D. degree in 1990. As a postdoctoral fellow, he worked with Professor Henry Taube at Stanford University from January 1991 to November 1992 and with Professor Robert Grubbs at California Institute of Technology from November 1992 to August 1994. In the fall of 1994, he was appointed as an assistant professor at North Dakota State University. He then moved to University of Virginia as an associate professor in the department of chemistry in 1997 and as a professor in 2003. The research projects in his laboratory focus on the design and synthesis of novel chiral molecules and macromolecules for applications in areas such as enantioselective fluorescent sensors, asymmetric catalysis, and electrical and optical materials.

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DEDICATION Dedicated to Professor Robert Grubbs at California Institute of Technology on the occasion of his 75th birthday. REFERENCES

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(24) (a) Yu, S.; Plunkett, W.; Kim, M.; Pu, L. Simultaneous Determination of Both the Enantiomeric Composition and Concentration of a Chiral Substrate with One Fluorescent Sensor. J. Am. Chem. Soc. 2012, 134, 20282−20285. (b) Yu, S.-S.; Plunkett, W.; Kim, M.; Wu, E.; Sabat, M.; Pu, L. Molecular Recognition of Aliphatic Diamines by 3,3′-Di(trifluoroacetyl)-1,1′-bi-2-naphthol. J. Org. Chem. 2013, 78, 12671−12680. (25) Wen, K. − L.; Yu, S. − S.; Huang, Z.; Chen, L. − M.; Xiao, M.; Yu, X. − Q.; Pu, L. Rational Design of a Fluorescent Sensor to Simultaneously Determine Both the Enantiomeric Composition and Concentration of Chiral Functional Amines. J. Am. Chem. Soc. 2015, 137, 4517−4524. (26) Wang, C.; Wu, E.; Wu, X. − D.; Xu, X. − X.; Zhang, G. − Q.; Pu, L. Enantioselective Fluorescent Recognition in the Fluorous Phase: Enhanced Reactivity and Expanded Chiral Recognition. J. Am. Chem. Soc. 2015, 137, 3747−3750. (27) Zeng, C. − Y.; Zhang, X. − L.; Pu, L. Enantioselective Fluorescence Imaging of Free Amino Acids in Living Cells. Chem. Eur. J. 2017, 23, 2432−2438. (28) Feagin, T. A.; Olsen, D. P.V; Headman, Z. C.; Heemstra, J. M. High-Throughput Enantiopurity Analysis Using Enantiomeric DNABased Sensors. J. Am. Chem. Soc. 2015, 137, 4198−4206. (29) Chandrasekhar, P.; Mukhopadhyay, A.; Savitha, G.; Moorthy, J. N. Remarkably selective and enantiodifferentiating sensing of histidine by a fluorescent homochiral Zn-MOF based on pyrene-tetralactic acid. Chem. Sci. 2016, 7, 3085−3091. (30) Akdeniz, A.; Minami, T.; Watanabe, S.; Yokoyama, M.; Ema, T.; Anzenbacher, P. Determination of enantiomeric excess of carboxylates by fluorescent macrocyclic sensors. Chem. Sci. 2016, 7, 2016−2022. (31) Shcherbakova, E. G.; Minami, T.; Brega, V.; James, T. D.; Anzenbacher, P. Determination of Enantiomeric Excess in Amine Derivatives with Molecular Self-Assemblies. Angew. Chem., Int. Ed. 2015, 54, 7130−7133. (32) Biedermann, F.; Nau, W. M. Noncovalent Chirality Sensing Ensembles for the Detection and Reaction Monitoring of Amino Acids, Peptides, Proteins, and Aromatic Drugs. Angew. Chem., Int. Ed. 2014, 53, 5694−5699. (33) Ito, H.; Shinoda, S. Chirality sensing and size recognition of NBoc-amino acids by cage-type dimeric lanthanide complexes: chirality detection of N-Boc-aspartate anions via luminescence colour change. Chem. Commun. 2015, 51, 3808−3811. (34) Song, F. Y.; Fei, N.; Zhang, S. W.; Cheng, Y. X.; Li, F.; Zhu, C. A chiral ionic polymer for direct visual enantioselective recognition of alpha-amino acid anions. Chem. Commun. 2013, 49, 2891−2893. (35) Iwaniuk, D. P.; Wolf, C. Chiroptical sensing of citronellal: systematic development of a stereodynamic probe using the concept of isostericity. Chem. Commun. 2012, 48, 11226−11228.

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