Free Amino Acid Recognition - American Chemical Society

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Free Amino Acid Recognition: A BisbinaphthylBased Fluorescent Probe with High Enantioselectivity Yuanyuan Zhu, Xue-Dan Wu, Shuang-Xi Gu, and Lin Pu J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b07803 • Publication Date (Web): 11 Dec 2018 Downloaded from http://pubs.acs.org on December 11, 2018

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Journal of the American Chemical Society

Free Amino Acid Recognition: A Bisbinaphthyl-Based Fluorescent Probe with High Enantioselectivity Yuan-Yuan Zhua,b, Xue-Dan Wub, Shuang-Xi Gub,*c and Lin Pu*b a

School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan 430205, P. R. China

b

Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA

c

Key Laboratory for Green Chemical Process of Ministry of Education, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430205, P. R. China *Corresponding author e-mail: [email protected], [email protected]

Abstract. A novel fluorescent probe based on a bisbinaphthyl structure has been designed and synthesized. This compound in combination with Zn(II) has exhibited highly enantioselective fluorescence enhancement with 13 common free amino acids. For example, its enantiomeric fluorescent enhancement ratios (ef or ∆IL/∆ID) in the presence of the following amino acids are extremely high: 177 for valine, 199 for methionine, 186 for phenylalanine, 118 for leucine, and 89 for alanine. The observed high enantioselectivity and the extent of the substrate scope are unprecedented in the fluorescent recognition of free amino acids. This fluorescent probe can be applied to determine the enantiomeric composition of the structurally diverse chiral amino acids. NMR and mass spectroscopic investigations have provided clues to elucidate the observed high enantioselectivity.

Introduction Development of molecular probes for enantioselective fluorescent recognition of chiral organic compounds has seen great progress in the past two decades.1-3 These probes have potential applications in high speed analysis of asymmetric reactions as well as monitoring chiral molecules in biological systems. Chiral amino acids are essential components of life and both enantiomers of their free forms have also displayed important biological functions.4-6 In addition, naturally occurring amino acids serve as versatile synthetic precursors to diverse functional organic compounds, and as chirality sources for asymmetric synthesis and catalysis.7.8 Enantioselective fluorescent recognition of amino acids has thus attracted significant research attention in recent years.1,9,10 However, many of the current fluorescent probes have exhibited only limited enantioselectivity in their fluorescent response toward free amino acids.1,9 Previously, we reported the 1,1’-bi-2-naphthol (BINOL)based compound (S)-1 as an enantioselective fluorescent probe in the presence of Zn(II) for the recognition of functional amines including free amino acids.11 When it was used to interact with phenylalanine, the enantioselectivity ef (ef = enantiomeric fluorescence enhancement ratio = [ILI0]/[ID-I0] = ∆IL/∆ID. I0: fluorescence intensity of the probe. IL or ID: fluorescence intensity with L or D substrate) was up to 13, but for other amino acids much lower enantioselectivity was observed (ef = 1 ~ 4). In order to further improve the enantioselectivity of (S)-1 in the fluorescent recognition of chiral amino acids, we have proposed to link two BINOLs together with a pyridine unit to CHO OH OH CHO (S)-1

design a new class of amino acid sensor and have discovered a highly enantioselective fluorescent probe with ef up to 199 for a number of free amino acids. Herein, these results are reported herein. Results and Discussion 1. Design and Synthesis of a BisBINOL-Pyridine-Based Molecular Probe Compound (S,S)-2 is designed for the recognition of amino acids in the presence of Zn(II). The two aldehyde groups of (S,S)-2 can react with the amine group of an amino acid and the central pyridine unit can increase the binding of a Zn(II) ion. The two chiral BINOL units have the potential to cooperate with each other to enhance the chiral bias when treated with the enantiomers of a chiral amino acid. For example, when (S,S)-2 is reacted with an amino acid and Zn2+, a complex like A could be generated from the condensation of the two aldehyde groups of the probe with the amine substrate followed by Zn(II) coordination. The matched and mismatched chiral configuration between the BINOL units and the amino acid units might lead to different fluorescence responses, giving the desired enantioselectivity. R CHO

OHC HO

OH O

N

(S,S)-2

O

OO *

R * N

N O O H H Zn O O L O O N

A

We have developed a quick synthesis of the newly designed bisBINOL compound as shown in Scheme 1. Reaction of the monoBINOL aldehyde (S)-3 with 2,6dibromomethylpyridine in the presence of a base at 50 oC gave compound (S,S)-4 in 73% yield. Removal of the MOM protecting groups of (S,S)-4 with HCl (conc.) led to the

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CHO

Br

CHO

N

N

Intensity (CPS)

O

DMF, 50 C MOM = CH3OCH2 conc. HCl CHCl3, ethanol reflux

ef = 176.5

5

8.0x10

(S,S)-4

5

4.0x10

0.0 500 550 600 650 Wavelength (nm)

6

1.6x10

1.2x106 8.0x105 4.0x105 0.0

(b) Intensity (CPS)

2.0x106

2.0x106 1.6x106 1.2x106 8.0x105 4.0x105

(S,S)-2 (S,S)-2 + 2eq Zn2+ + 0.5 eq D-valine + 1 eq D-valine + 2 eq D-valine 2x104 + 3 eq D-valine + 4 eq D-valine 1x104 + 5 eq D-valine 0 + 6 eq D-valine + 8 eq D-valine + 10 eq D-valine

500 550 600 Wavelength (nm)

650

700

450

Intensity (CPS)

1.6x106 1.2x106 8.0x105

0.0 6.5

7.0

7.5 8.0 pH value

8.5

9.0

Figure 2. Fluorescence intensity of (S,S)-2 (0.01 mM in CH3CN, 1.0 equiv) with L- or D-valine (5.0 equiv, in buffer of various pHs) in the presence of Zn(OAc)2 (in H2O, 2.0 equiv) at 522 nm versus the pH (pH 6.0 buffer: 0.1 M AcOH-AcONa. pH 7.5 and 8.2 buffer: 25 mM HEPES. pH 8.8 buffer: 25 mM BICINE. CH3CN/H2O = 99/1, v/v. λexc = 435 nm. Slit: 3/3 nm).

500 600 700

500 550 600 650 Wavelength/nm

0 1 2 3 4 5 6 7 8 9 10 11 Equivalent of valine

4.0x105

0.0 450

700

(S,S)-2 (S,S)-2+2eq Zn2+ +5 eq D-valine +5 eq L-valine

2.0x106

6.0

(a)

+ 0~10 eq D-valine + 0~10 eq L-valine

8.0x105

The enantiomer of (S,S)-2, compound (R,R)-2, was prepared from the (R)-BINOL-based precursor and its fluorescence response toward D- and L-valine was studied under the same conditions. As shown in Figure S2, a mirrorimage relation was observed between the fluorescence responses of (R,R)-2 and (S,S)-2 toward the enantiomers of the valine which confirms the inherent chiral recognition process. The above fluorescence measurements demonstrate that the bisBINOL-based probe (S,S)-2 exhibits greatly enhanced enantioselectivity over the monoBINOL compound (S)-1 in the fluorescent recognition of the amino acid. It has validated the proposed bisBINOL-pyridine strategy. In the fluorescence measurements of Figure 1, the amino acid valine was in its deprotonated form in the BICINE buffer solution at pH = 8.8 before it was treated with the sensor. We have studied the effect of varying the initial pHs of the amino acid on the fluorescence response of (S,S)2+Zn(OAc)2. As shown in Figure 2, at pH = 7.5, both fluorescence enhancement and enantioselectivity were low. However, under either the acidic (pH = 6) or basic conditions (pH = 8.8), L-valine greatly enhanced the fluorescence with high enantioselectivity. This is consistent with the reaction of the probe with the chirality matched substrate by either the acid or base catalysed amine-aldehyde condensation to form imines.

(S,S)-2

2. Fluorescent Responses toward L- and D-Valine Previously, when the monoBINOL compound (S)-1 was used to interact with L- and D-valine in the presence of Zn(OAc)2, the observed ef was only about 1.5.11 That is, the enantioselective fluorescent response of (S)-1 is very low toward this amino acid. In order to test the ability of the new bisBINOL compound in the fluorescent recognition of free amino acids, the fluorescent response of (S,S)-2 toward the two enantiomers of valine in the presence of Zn(OAc)2 was first studied. When (S,S)-2 (0.01 mM, CH3CN) was combined with Zn(OAc)2 (2 equiv), little change was observed in the fluorescence. As shown in Figure 1a, when L-valine (0.5 – 10 equiv, BICINE pH = 8.8) was added, there was large fluorescence enhancement at λ = 522 nm. When D-valine was used, however, there was very low fluorescence enhancement under the same conditions (Figure 1b). We examined how the reaction time could affect the fluorescence response. As shown in Figure S1, the fluorescence enhancement of (S,S)-2+Zn(OAc)2 with Lvaline (5 equiv) became stable after 3 h but little fluorescence enhancement was observed with the addition of D-valine over 5 h. We thus chose to carry out the fluorescence measurement after 3 h of the reaction even though this may or may not be when the reaction of the probe with the substrate reaches equilibrium. Figure 1c compares the fluorescence responses of (S,S)-2+Zn(OAc)2 with L- and D-valine (5 equiv) which gave ef [(IL-I0)/(ID-I0)] at 177. In Figure 1d, the fluorescence intensity at 522 nm is plotted versus the amount of valine which demonstrates that the enantioselective fluorescent response of (S,S)-2 reached maximum at 5 equiv valine and then maintained at a similar level when the amount of the amino acid further increased. (S,S)-2 (S,S)-2 + 2eq Zn2+ + 0.5 eq L-valine + 1 eq L-valine + 2 eq L-valine + 3 eq L-valine + 4 eq L-valine + 5 eq L-valine + 6 eq L-valine + 8 eq L-valine + 10 eq L-valine

1.2x106

Figure 1. Fluorescence spectra of (S,S)-2 (0.01 mM in CH3CN, 1.0 equiv) with (a) L-valine or (b) D-valine (in pH 8.8 BICINE buffer, 0.5~10 equiv) in the presence of Zn(OAc)2 (in H2O, 2.0 equiv) (CH3CN/H2O = 99/1, v/v). (c) Fluorescence response of (S,S)-2+Zn(OAc)2 (2.0 equiv) toward L- and D-valine (5.0 equiv). (d) The Fluorescence intensity at 522 nm versus the equivalent of L- and D-valine (The error bars from three independent experiments. λexc = 435 nm. Slit: 3/3 nm).

o

(S)-3

1.2x106

450

OHC

O

K2CO3

1.6x106

0.0

OMOM MOMO

OMOM OH

2.0x106

4.0x105

Scheme 1. Synthesis of the BisBINOL Dialdehyde (S,S)-2. Br

(d) (S,S)-2 2.0x106 (S,S)-2+2eq Zn2+ +5eq D-valine 1.6x106 +5eq L-valine

(c)

Intensity (CPS)

formation of the desired product (S,S)-2 in 91% yield. In the 1 H NMR spectrum of (S,S)-2 in CDCl3, its hydroxyl signal was found to be a singlet at δ 10.47 (2H). This is similar to that of the hydroxyl groups in (S)-1, indicating similar intramolecular hydrogen bonding in these compounds. The 1 H NMR spectrum of (S,S)-2 also demonstrates that it maintains C2 symmetry in solution. This compound is found to be nonfluorescent in CH3CN solution.

Intensity (CPS)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

700

We studied the interaction of both (S,S)- and (R,R)-2 with valine at various enantiomeric composition, and plotted the fluorescence response of each enantiomeric probe versus the enantiomeric excess [ee = ([L]-[D])/([L]+[D])] of valine in Figure 3. The mirror-image relation was observed between the

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Intensity (CPS)

2.5x106

(S,S)-2 (R,R)-2

2.0x106

4. Study of the Interaction of (S,S)-2+Zn(OAc)2 with Valine by NMR and Mass Spectroscopic Analyses. We conducted a 1H NMR spectroscopic study for the reaction of (S,S)-2+Zn(OAc)2 with L- and D-valine. (S,S)-2 was dissolved in CD3CN (250 µL, 0.2 mM). The Zn(OAc)2 solution and BICINE buffer (25 mM, pH 8.8) were prepared by using D2O as the solvent. As shown in Figure 5a, (S,S)-2 gave two singlets with one at δ 10.17 for its aldehyde protons and another at δ 8.49 for the naphthyl proton ortho to the aldehyde groups. When Zn(OAc)2 was added, no change was observed. Then, addition of L-valine led to the appearance of a new aldehyde signal at δ 10.15. In addition, two new signals at δ 8.60 and 8.47 were observed which can be assigned to an imine proton and the naphthyl proton ortho to the newly formed imine group from the condensation of one aldehyde group of (S,S)-2 with the amine group of valine. As shown in Figure 5a, at the equilibrium for the reaction of (S,S)-2 with L-valine in the presence of Zn(OAc)2, the unreacted (S,S)-2 predominated. Figure 5b shows the 1H NMR spectra for the reaction of (S,S)-2+Zn(OAc)2 with D-valine. Only one new signal at δ 8.60 was observed which could be assigned to an imine proton produced from the amine-aldehyde reaction. The shift of both the aldehyde proton and the ortho-aromatic proton signals was not obvious. In this experiment, (S,S)-2 also predominated after 2 d reaction. In the upfield signal region, two new doublets at δ 0.8-1.2 were observed from the two diastereomeric methyl groups of L- or D-valine in both experiments, indicating formation of new compounds from the reaction of (S,S)-2 with both L- and D-valine [Figure S5 (a) and (b)].

1.5x106 1.0x106 5.0x105 0.0 -100-80 -60 -40 -20 0 20 40 60 80 100 ee of L-valine

Figure 3. Fluorescence intensity of (S,S)-2 and (R,R)-2 (0.01 mM in CH3CN, 1 equiv) at 522 nm versus the ee value of L-valine (in pH 8.8 BICINE buffer, 5 equiv) in the presence of Zn(OAc)2 (in H2O, 2.0 equiv) (The error bars from three independent experiments. λexc = 435 nm. Slit: 3/3 nm). 3. Fluorescent Responses toward Additional Amino Acids We studied the fluorescence responses of (S,S)-2 in combination with Zn(OAc)2 toward 18 common amino acids (including valine). Under the same conditions, enantioselective fluorescence enhancements were observed for the following 13 amino acids: valine, methionine, phenylalanine, leucine, alanine, tryptophan, glutamine, tyrosine, asparagine, threonine, serine, histidine and arginine. In general, while the D-enantiomers of these amino acids cannot significantly increase the fluorescence of the probe, the L-enantiomers greatly enhance the fluorescence at above 500 nm (Figure S3). Extremely high ef values of (S,S)-2 in the presence of 5 amino acids were observed with 177 for valine, 199 for methionine, 186 for phenylalanine, 118 for leucine and 89 for alanine, respectively. Figure 4 gives the fluorescence responses of (S,S)-2+Zn(OAc)2 toward the enantiomers of methionine, phenylalanine, leucine and alanine which exhibit an essentially on/off enantioselectivity, that is, one enantiomer turns on the fluorescence of the probe and another enantiomer maintains it at the off state. (S,S)-2 also showed high ef values with the following 6 amino acids: tryptophan (38), glutamine (29), tyrosine (25), asparagine (18), threonine (10) and serine (8)

Intensity (CPS)

1.6x106 Slit = 3/3 nm 1.2x106 8.0x105

(S,S)-2 (S,S)-2+2eq Zn2+ +5eq D-methionine +5eq L-methionine ef = 199.4

5

(b) Intensity (CPS)

(a)

3

was found with proline, aspartic acid, glutamic acid, cysteine and lysine (Figure S3). The observed extremely high enantioselectivity and the extent of the substrates scope for the use of (S,S)-2+Zn(OAc)2 are unprecedented in the fluorescent recognition of free amino acids.

fluorescence responses of this enantiomeric probe pair. These plots can be used to determine the enantiomeric composition of the amino acid.

4.0x10

3.5x106 Slit = 3/3 nm 2.8x106

(S,S)-2 (S,S)-2+2eq Zn2+ +5eq D-phenylalanine +5eq L-phenylalanine

2.1x106 ef = 185.9 1.4x106 5

7.0x10

0.0

0.0

(c) 4.0x106

500 550 600 650 Wavelength (nm)

Slit = 5/5 nm

6

3.2x10

2.4x106 1.6x106

700

(S,S)-2 (S,S)-2+2eq Zn2+ +5eq D-leucine +5eq L-leucine ef = 117.5

8.0x105 0.0

450

(d)

500 550 600 Wavelength (nm)

3.0x106 Slit = 5/5 nm

Intensity (CPS)

450

Intensity (CPS)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2.4x10

1.8x106

650

700

(S,S)-2 (S,S)-2+2eq Zn2+ +5eq D-alanine +5eq L-alanine

Figure 5. 1H NMR spectra of the reaction mixture of (S,S)-2 (500 µL, 0.2 mM in CD3CN, 1.0 equiv) with (a) L-valine or (b) Dvaline (50 μL, 16 mM in BICINE buffer, 8.0 equiv) in the presence of Zn(OAc)2 (50 μL, 4 mM in D2O, 2.0 equiv) at various reaction time. For both plots: 1, (S,S)-2+buffer. 2, (S,S)2+buffer+2 equiv Zn(II). 3 - 7, +L- or D-valine for 1 h, 3 h, 5 h, 8 h and 2 d respectively (BICINE buffer: 25 mM at pH 8.8 made with D2O. The other signals of (S,S)-2, valine and BICINE at δ 08 and the vacant region at δ 8.9-9.8 are removed for clarity. The full spectra are given in Figure S5).

ef = 89.4

1.2x106 5

6.0x10

0.0

450

500 550 600 650 Wavelength/nm

700

450

500 550 600 650 Wavelength (nm)

700

Figure 4. Fluorescence response of (S,S)-2 (0.01 mM in CH3CN, 1.0 equiv) toward (a) methionine, (b) phenylalanine, (c) leucine and (d) alanine (in pH 8.8 BICINE buffer, 5 equiv) in the presence of Zn(OAc)2 (in H2O, 2.0 equiv) (CH3CN/H2O = 99/1, v/v. λexc = 435 nm). (Figure S4). Moderate enantioselectivity with histidine and arginine was observed (Figure S4). Little fluorescence response

The above study demonstrates that even though only small portion of (S,S)-2 reacts with L-valine in the presence of Zn(OAc)2 in CD3CN, the resulting product has generated the observed greatly enhanced fluorescence. In sharp contrast, although D-valine reacts with (S,S)-2+Zn(OAc)2 to a similar

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extent as L-valine, the resulting product gives little fluorescence, indicating a very different structure. We obtained the mass spectra (MALDI-TOF) for the above reaction mixtures of (S,S)2+Zn(OAc)2 with L- and D-valine, but only the peaks corresponding to that of (S,S)-2 was observed in both cases with little information that could be extracted for the products (Figure S6). In order to gain more information for the reaction of (S,S)-2 +Zn(OAc)2 with L- and D-valine, we conducted 1H NMR spectroscopic study in DMSO-d6 under the same conditions as in CD3CN. The reactions proceeded to a greater extent than those in CD3CN and the unreacted (S,S)-2 appeared to be less than the products after 2 d (Figure S8). We also observed that both L- and D-valine greatly enhanced the fluorescence intensity of (S,S)2+Zn(OAc)2 when the reaction was conducted in DMSO with little enantioselectivity (Figure S7). The mass spectra (MALDI-TOF) of the reaction mixtures of (S,S)-2+Zn(OAc)2 with L- and D-valine in DMSO were obtained (Figure S9). In the mass spectrum for the reaction of (S,S)2+Zn(OAc)2 with L-valine, a predominate signal at m/z = 1027.5 was observed which could be attribute to that of 5 (calcd for 5: 1027.3), an analog of the proposed complex A. Figure 6 shows a proposed structure for this complex and the molecular model was obtained by using the Spartan program (Semi-empirical calculation-PM3). In the mass spectrum (Figure S9) obtained for the reaction of D-valine, the base peak at m/z = 831.5 can be attributed to the molecular ion of 6 (calcd for 6+H: 831.3). This demonstrates that L-valine and D-valine favor the formation of different products and it is much more difficult for D-valine to generate the double condensation product like 5. Thus, the reaction of (S,S)-2 with second equivalent of D-valine is not as favorable as it with Lvaline probably due to either greater steric hindrance in the

O O HO

O N O

N

O

L Zn

H N

O

H O N

H

OO

5

L = H2O

HO N

OH

H O N

OH2 N

O

N O

OHC

OH O

Experiment Section General Data. Nitrogen atmosphere was applied to all the synthetic reactions unless otherwise noted and the commercially available compounds were from Sigma Aldrich Chemical Co. or Alfa Aesar. All solvents used in the fluorescent measurement were HPLC grades. NMR spectra were recorded on a Varian-600 MHz spectrometer. Optical rotation measurements were conducted on a Jasco P-2000 digital polarimeter. Chemical shifts for 1H NMR spectra were recorded in parts per million relative to solvent signals at 7.26 ppm for CDCl3, 1.94 ppm for CD3CN, and 2.50 ppm for DMSO-d6. Chemical shifts for 13C NMR were recorded relative to the centerline of a triplet at 77.16 ppm for CDCl3. Mass spectroscopic analyses were conducted by the University of Illinois at Urbana-Champaign Mass Spectrometry Facility. The steady-state fluorescence emission spectra were recorded with a Horiba FluoroMax-4 spectrofluorometer.

CHO O

O

Zn

O

Conclusion A new bisBINOL-based dialdehyde has exhibited unprecedented highly enantioselective fluorescent enhancement toward structurally diverse amino acids in the presence of Zn(II). This compound can be used as a fluorescent probe to determine the enantiomeric composition of various free amino acids. It is proposed that the cooperation of the two BINOL units as well as the linking pyridine unit might have greatly enhanced the chiral bias in the reaction of the probe with the amino acid enantiomers, leading to the highly enantioselective fluorescent response. Formation of the structurally rigid zinc complexes like 5 could be used to account for the enhanced fluorescence generated from the chirality matched probe-substrate interaction.

O

H

H2O

H

reaction of 6 with D-valine or lower stability of the D-valine derived product like 5. In the mass spectrum of (S,S)2+Zn(OAc)2 with either L- or D-valine, a peak at m/z = 1758.9 is identified as the dimeric compound 7 (calcd for 7: 1758.5) formed from the coordination of two molecules like 6 with a Zn2+. The mass spectroscopic study for the reaction of (S,S)2+Zn(OAc)2 with L- and D-valine in DMSO has provided the evidence for the different reactivity of the two enantiomeric amino acids with the chiral probe although the fluorescence response difference is not significant in this solvent system. We thus propose that when (S,S)-2+Zn(OAc)2 is treated with the enantiomeric amino acids in CH3CN, there should also be different product distribution, generating the observed highly enantioselective fluorescent response.

O

L

H O

O

N

7

Figure 6. Proposed Reaction Products 5, 6 and 7. O

CHO

Synthesis and Characterization of (S,S)-4. A mixture of (S)-3 (507 mg, 1.42 mmol), bis(bromomethyl)pyridine (150 mg, 0.57 mmol) and K2CO3 (312 mg, 2.26 mmol) in N,Ndimethyformamide (5 mL) was heated at 50 oC for 15 h. Then the mixture was poured into H2O (40 mL) at room temperature, and extracted with EtOAc (2×30 mL). The resulting organic solution was washed with H2O (1×40 mL) and brine (2×40 mL), and dried with anhydrous Na2SO4. After filtration, the solvent of the

O

6

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Journal of the American Chemical Society organic layer was removed under vacuum and purification was conducted by column chromatography on silica gel eluted gradiently with 20-40% EtOAc in hexane to afford (S,S)-4 as a yellow solid in 73% yield (340 mg). 1H NMR (600 MHz, CDCl3) δ 10.57 (s, 2H), 8.57 (s, 2H), 8.04 (d, J = 8.2 Hz, 2H), 7.97 (d, J = 9.1 Hz, 2H), 7.88 (d, J = 8.1 Hz, 2H), 7.46-7.40 (m, 4H), 7.37 (dd, J = 8.2, 6.7 Hz, 2H), 7.33-7.28 (m, 4H), 7.20 (dd, J = 8.6, 4.5 Hz, 4H), 7.04 (t, J = 7.8 Hz, 1H), 6.51 (d, J = 7.8 Hz, 2H), 5.14 (q, J = 13.7 Hz, 4H), 4.71 (d, J = 5.9 Hz, 2H), 4.62 (d, J = 5.9 Hz, 2H), 2.93 (s, 6H). 13C NMR (150 MHz, CDCl3) δ 191.27, 156.44, 154.01, 153.91, 137.27, 137.10, 133.89, 131.25, 130.56, 130.33, 130.28, 129.36, 129.23, 129.15, 128.22, 127.25, 126.93, 126.06, 125.21, 124.29, 119.52, 118.79, 114.60, 100.37, 71.50, 57.25.

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Lin Pu: 0000-0001-8698-3228 Supplementary Materials Available: Additional experimental description and spectroscopic data. Keywords: amino acid; enantioselectivity; fluorescence; chiral recognition; BINOL References 1. (a) Pu, L. Fluorescence of Organic Molecules in Chiral Recognition. Chem. Rev. 2004, 104, 1687–1716. (b) Accetta, A.; Corradini, R.; Marchelli, R. Enantioselective Sensing by Luminescence. Top. Curr. Chem. 2011, 300, 175−216. (c) Zhang, X.; Yin, J.; Yoon, Juyoung. Recent Advances in Development of Chiral Fluorescent and Colorimetric Sensors. Chem. Rev. 2014, 114, 4918–4959. (d) You, L.; Zha, D.; Anslyn, E. V. Recent Advances in Supramolecular Analytical Chemistry Using Optical Sensing. Chem. Rev. 2015, 115, 7840–7892. 2. (a) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Chiral Discrimination of Monosaccharides Using a Fluorescent Molecular Sensor. Nature 1995, 374, 345–347. (b) Pugh, V.; Hu, Q. -S.; Pu, L. The First Dendrimer-Based Enantioselective Fluorescent Sensor for the Recognition of Chiral Amino Alcohols. Angew. Chem. Int. Ed. 2000, 39, 3638–3641. (c) Lin, J.; Hu, Q. -S.; Xu, M. -H.; Pu, L. A Practical Enantioselective Fluorescent Sensor for Mandelic Acid. J. Am. Chem. Soc. 2002, 124, 2088–2089. (d) Zhao, J.; Fyles, T. M.; James, T. D. Chiral Binol–Bisboronic Acid as Fluorescence Sensor for Sugar Acids. Angew. Chem., Int. Ed. 2004, 43, 3461–3464. (e) Zhu, L.; Anslyn, E. V. Facile Quantification of Enantiomeric Excess and Concentration with Indicator-Displacement Assays: An Example in the Analyses of α-Hydroxyacids. J. Am. Chem. Soc. 2004, 126, 3676–3677. (f) Mei, X.; Wolf, C. Enantioselective Sensing of Chiral Carboxylic Acids. J. Am. Chem. Soc. 2004, 126, 14736– 14737. 3. (a) Pu, L. Enantioselective Fluorescent Sensors: A Tale of BINOL. Acc. Chem. Res. 2012, 45, 150–163. (b) Pu, L. Simultaneous Determination of Concentration and Enantiomeric Composition in Fluorescent Sensing. Acc. Chem. Res. 2017, 50, 1032–1040. 4. (a) Holden, J. T. Amino Acid Pools. Distribution, formation and function of free amino acids. Elsevier, Amsterdam. 1962. (b) Lubec, C. Amino Acids (Chemistry, Biology, Medicine). Escom New York. 1990. 5. Nefyodov L. Amino Acids and Their Derivatives (chemistry, biochemistry, pharmacology, medicine). Proc of Internat. Symp; Grodno. 1996. 6. (a) Konno, R.; Brückner, H.; D’Aniello, A.; Fisher, G.; Fujii, N.; Homma, H. (Eds) D-Amino Acids: A New Frontier in Amino Acids and Protein Research - Practical Methods and Protocols. Nova Science, New York, 2007. (b) Weatherly, C. A.; Du, S.; Parpia, C.; Santos, P. T.; Hartman, A. L.; Armstrong, D. W. DAmino Acid Levels in Perfused Mouse Brain Tissue and Blood: A Comparative Study. ACS Chem. Neurosci. 2017, 8, 1251–1261. 7. (a) List, B.; Lerner, R. A.; Barbas III, C. F. Proline-Catalyzed Direct Asymmetric Aldol Reactions. J. Am. Chem. Soc. 2000, 122, 2395–2396. (b) MacMillan, D. W. C. The Advent and Development of Organocatalysis. Nature 2008, 455, 304–308. (c) Smith, J. M.; Qin, T.; Merchant, R. R.; Edwards, J. T.; Malins, L. R.; Liu, Z.; Che, G.; Shen, Z.; Shaw, S. A.; Eastgate, M. D.; Baran, P. S. Decarboxylative Alkynylation. Angew. Chem. Int. Ed. 2017, 56, 11906–11910. 8. Amino acid-based chiral ligands: Micskei, K.; Patonay, T.; Caglioti, L.; Pályi, G. Amino Acid Ligand Chirality for Enantioselective Syntheses. Chem. Biodiversity 2010, 7, 16601669. 9. Reports on enantioselective fluorescent recognition of free amino acids (not including N- or O-protected amino acid derivatives): (a) Pagliari, S.; Corradini, R.; Galaverna, G.; Sforza, S.; Dossena, A.; Montalti, M.; Prodi, L.; Zaccheroni, N.; Marchelli, R.

Synthesis and Characterization of (S,S)-2. Conc. HCl (2 mL) and ethanol (1 mL) were added to (S,S)-4 (205 mg, 0.25 mmol) in CHCl3 (1 mL) at rt. After heated at reflux for 6 h, the reaction mixture was poured into H2O (10 mL) at rt and neutralized with saturated NaHCO3 solution until no gas was evolved. It was then extracted with EtOAc (2×20 mL) and concentrated under reduced pressure to give the crude product in 99% yield (181 mg, yellow solid). It was further purified by column chromatography on silica gel eluted gradiently with 25-50% ethyl acetate in hexane which gave (S,S)-2 in 91% yield (166 mg, yellow solid). 1H NMR (600 MHz, CDCl3) δ 10.47 (s, 2H), 10.17 (s, 2H), 8.29 (s, 2H), 7.97-7.86 (m, 6H), 7.38-7.19 (m, 14H), 7.11 (t, J = 7.8 Hz, 1H), 6.67 (d, J = 7.7 Hz, 2H), 5.17 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 196.82, 156.69, 153.84, 153.59, 137.99, 137.86, 137.27, 133.76, 130.47, 130.31, 129.85, 129.59, 128.35, 127.65, 126.92, 125.51, 124.99, 124.44, 124.13, 122.24, 119.52, 118.68, 118.10, 115.08, 71.40. HRMS Calcd for C49H34NO6 (MH+): 732.2386, Found: 732.2372 (TOF-MS). [α]23 D = -109.8 (c = 1.0, CHCl3). Synthesis and Characterization of (R,R)-2. Compound (R,R)-2, the enantiomer of (S,S)-2, was obtained as a yellow solid in a twostep yield of 71% by using the same procedure as described above but starting with (R)-3. 1H NMR (600 MHz, CDCl3) δ 10.48 (s, 2H), 10.17 (s, 2H), 8.29 (s, 2H), 7.97-7.87 (m, 6H), 7.39-7.18 (m, 14H), 7.11 (t, J = 7.8 Hz, 1H), 6.67 (d, J = 7.7 Hz, 2H), 5.17 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 196.81, 156.74, 153.86, 153.59, 137.98, 137.85, 137.17, 133.76, 130.45, 130.28, 129.85, 129.58, 128.34, 127.63, 126.91, 125.49, 124.98, 124.42, 124.11, 122.24, 119.47, 118.67, 118.09, 115.08, 71.48. HRMS Calcd for C49H34NO6 (MH+): 732.2386, Found: 732.2405 (TOF-MS). [α]23 D = +110.7 (c = 1.0, CHCl3). Sample Preparation for Fluorescence Measurement. Stock solutions of 0.2 mM (S,S)-2 or (R,R)-2 in CH3CN or DMSO, 4 mM Zn(OAc)2 in H2O, and 1 – 20 mM amino acid in pH 8.8 BICINE buffer were all freshly prepared for each measurement. The reaction mixtures were all allowed to stand at rt for 3 h (unless otherwise noted) without nitrogen protection. Then, the reaction mixtures were diluted to the desired concentration of 0.01 mM with CH3CN or DMSO. Fluorescence measurements were conducted after 1 h, and finished within 30 min. Acknowledgement Partial supports from the National Science Foundation of US (CHE-1565627), the Chinese National Natural Science Foundation (Nos. 21877087, 21602164), Wuhan Institute of Technology Scientific Research Fund (No. K201716), and Wuhan International Scientific and Technological Cooperation Project (No. 2017030209020257) are gratefully acknowledged. YYZ also thanks the China Scholarship Council (No. 201608420202). ORCID Yuan-Yuan Zhu: 0000-0003-4526-5048 Shuang-Xi Gu: 0000-0003-0159-4616

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Enantioselective Fluorescence Sensing of Amino Acids by Modified Cyclodextrins: Role of the Cavity and Sensing Mechanism. Chem. Eur. J. 2004, 10, 2749–2758. (b) Corradini, R.; Paganuzzi, C.; Marchelli, R.; Pagliari, S.; Sforza, S.; Dossena, A.; Galaverna, G.; Duchateau, A. Fast Parallel Enantiomeric Analysis of Unmodified Amino Acids by Sensing with Fluorescent β-Cyclodextrins. J. Mater. Chem. 2005, 15, 2741– 2746. (c) Dai, Z.; Xu, X.; Canary, J. W. Rigidified Tripodal Chiral Ligands in the Asymmetric Recognition of Amino Compounds. Chirality 2005, 17, S227–S233. (d) Wang, H.; Chan, W. H.; Lee, A. W. M. Cholic Acid-Based Fluorescent Probes for Enantioselective Recognition of Trifunctional Amino Acids. Org. Biomol. Chem. 2008, 6, 929–934. (e) Su, X.; Luo, K.; Xiang, Q.; Lan, J.; Xie, R. Enantioselective Recognitions of Chiral Molecular Tweezers Containing Imidazoliums for Amino Acids. Chirality 2009, 21, 539–546. (f) Kwong, H. L.; Wonga, W. L; Lee, C. S.; Yeung, C. T.; Teng, P. F. Zinc(II) Complex of Terpyridine-Crown Macrocycle: A New Motif in Fluorescence Sensing of Zwitterionic Amino Acids. Inorg. Chem. Commun. 2009, 12, 815–818. (g) Yang, L.; Qin, S.; Su, X.; Yang, F.; You, J.; Hu, C.; Xie, R.; Lan, J. 1,1'-Binaphthyl-based Imidazolium Chemosensors for Highly Selective Recognition of Tryptophan in Aqueous Solutions. Org. Biomol. Chem. 2010, 8, 339–348. (h) Tang, L.; Wei, G.; Nandhakumar, R.; Guo, Z. Facile Synthesis of the Uryl Pendant Binaphthol Aldehyde and Its Selective

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Fluorescent Recognition of Tryptophan. Bull. Korean Chem. Soc. 2011, 32, 3367–3371. (i) Li, F.; Li, L.; Yang, W.; Zheng, L.-S.; Zheng, Z.-J.; Jiang, K.; Lu, Y.; Xu, L.-W. Chiral Ar-BINMOLDerived Salan as Fluorescent Sensor for Recognition of CuCl and Cascade Discrimination of α-Amino Acids. Tetrahedron Lett. 2013, 54, 1584–1588. (j) Feng, H.-T.; Zhang, X.; Zheng, Y.-S. Fluorescence Turn-on Enantioselective Recognition of Both Chiral Acidic Compounds and α-Amino Acids by a Chiral Tetraphenylethylene Macrocycle Amine. J. Org. Chem. 2015, 80, 8096–8101. (k) Wang, C.; Zeng, C.; Zhang, X.; Pu, L. Enantioselective Fluorescent Recognition of Amino Acids by Amide Formation: An Unusual Concentration Effect. J. Org. Chem. 2017, 82, 12669–12673. (l) Zeng, C.; Zhang, X.; Pu, L. Enantioselective Fluorescent Imaging of Free Amino Acids in Living Cells. Chem. Eur. J. 2017, 23, 2432–2438. 10. A review on fluorescent detection of amino acids without enantioselective recognition: Zhou, Y.; Yoon, J. Recent Progress in Fluorescent and Colorimetric Chemosensors for Detection of Amino Acids. Chem. Soc. Rev. 2012, 41, 52–67. 11. Huang, Z ;Yu, S. ; Wen, K.; Yu，X.; Pu, L. Zn(II) Promoted Dramatic Enhancement in the Enantioselective Fluorescent Recognition of Functional Chiral Amines by a Chiral Aldehyde. Chem. Sci. 2014, 5, 3457–3462.

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