1. Introduction

Chirality, a ubiquitous property in nature, plays a critical role in biochemistry and life processes. The 20 common α-Amino acids (except glycine) are typical chiral molecules that occur as L- and D-enantiomers. Despite their structural similarity, the difference in their spatial configuration results in profound disparities in biological activity^[1-3]. L-amino acids are the basic building blocks of proteins, essential for cellular metabolism, signal transduction, and physiological function maintenance^[4,5]. Organisms predominantly use L-amino acids for protein synthesis, they form protein backbones, guide proper protein folding, and underpin key applications^[6-9]. L-tryptophan (L-Trp) aids antidepressant and sleep drugs^[10,11], L-glutamine (L-Gln) supports clinical nutrition^[12,13], L-glutamic acid (L-Glu) can participate in the tricarboxylic acid cycle, providing energy for cells^[14,15], and L-alanine (L-Ala) can participate in the glucose-alanine cycle, transporting energy between muscles and the liver^[16,17]. Once considered “non-natural”, D-amino acids have been increasingly identified to be widely distributed in organisms, either in free form or within peptides, where they serve diverse biological functions^[18]. Examples of their functions include: D-Ala and D-Glu form bacterial cell wall peptidoglycan^[19,20]; β-lactam antibiotics such as penicillin exert antibacterial effects by mimicking the D-Ala-D-Ala motif to inhibit cell wall cross-linking^[21,22]; D-serine (D-Ser) is critically involved in the regulation of learning and memory, and its aberrant levels are closely associated with schizophrenia and Alzheimer’s disease^[23-25]; similarly, D-aspartate (D-Asp) also shows significant expression alterations in the prefrontal cortex of patients suffering from these two neurodegenerative and psychiatric disorders^[26]. Thus, L- and D-amino acids play divergent roles in biological processes, reflecting the precision and complexity of the chiral world. Efficient enantioselective recognition and analysis of amino acids are vital for exploring their functions and investigating disease pathogenesis^[27].

Enantiomeric recognition of amino acids also governs the safety and efficacy of chiral drugs, primarily because biological targets such as receptors and enzymes are constructed from chiral amino acids and exhibit inherent stereoselectivity. In most cases, only one enantiomer can properly bind to the target and produce the desired therapeutic effect, whereas its antipode may be inactive, weakly active, or even toxic and cause adverse reactions. Moreover, chiral recognition also affects drug absorption, metabolism, and elimination, leading to significant differences in pharmacokinetic profiles between enantiomers. Therefore, enantioselective recognition of amino acids is also of great significance for the development of chiral drugs^[28-30].

Compared with traditional chiral analysis techniques such as high-performance liquid chromatography^[31-33], gas chromatography^[34,35], capillary electrophoresis^[36-38], and ¹H nuclear magnetic resonance spectroscopy (¹H NMR)^[39-42], fluorescent sensing has emerged as a promising alternative for chiral analysis due to its advantages of high sensitivity, rapid response, as well as unique visualization and in-situ detection capabilities^[43-45]. Commonly used chiral fluorescent analytical techniques include chiral fluorescent crown ether probes, chiral quantum dot-based fluorescent probes, chiral polymer fluorescent probes, metal organic framework (MOF) and covalent organic framework (COF)-based fluorescent probes, and host-guest inclusion-assembled fluorescent sensing systems. Certainly, modern chiral analysis techniques are advancing rapidly, with various emerging methods featuring unique characteristics and gradually forming complementary advantages. For instance, compared with the emerging ¹⁹F NMR chiral analysis methods^[46-48], fluorescent probe approaches are less competitive in terms of anti-interference toward background signals and signal assignment for simultaneous detection of multiple analytes. Nevertheless, they exhibit distinct advantages in high-throughput rapid screening and in situ real-time imaging.

The core of the fluorescent probe technology lies in the design and synthesis of novel chiral fluorescent probes that can specifically bind to target enantiomers and generate differential fluorescent signal responses. In the field of chiral fluorescent recognition of amino acids, Pu^[49] has made pioneering contributions, particularly in the development of fluorescent probes based on axially chiral (S)/(R)-1,1’-bi-2-naphthol (BINOL) scaffolds. In 2014, Pu’s group^[50] reported that Zn²⁺ could amplify the enantioselective fluorescent response of (S)/(R)-3,3’-diformyl-BINOL probes toward chiral amines, including amino acids. Subsequently, the group has achieved breakthrough progress in the simultaneous determination of total concentration and optical composition of enantiomeric mixtures^[51], the design of pseudo-enantiomers^[52], high enantioselectivity^[53], the construction of near-infrared fluorescent probes^[54], chiral recognition in aqueous media^[55], and fluorous phases^[56].

Besides BINOL-based chiral probes, cyclodextrin-based and calixarene-based chiral probes have also attracted considerable attention. Cyclodextrin-based chiral fluorescent probes^[57,58] feature a unique cavity structure that forms host–guest inclusion complexes, giving them high selectivity toward specific chiral analytes, together with good water solubility and biocompatibility favorable for aqueous and biological applications. However, their fixed cavity size limits applicability to analytes of varying dimensions, and their typically weak fluorescence response and low quantum yield compromise detection sensitivity. Calixarene-based chiral probes^[59,60] show outstanding structural tunability; their cavity size and binding sites can be readily modified to accommodate diverse chiral analytes. Nonetheless, their synthesis is often complex and low-yielding, driving up preparation costs and limiting their application.

Among numerous fluorescent probe scaffolds, Schiff base structures exhibit significant advantages, including simple synthesis, flexible structural tunability, excellent coordination ability, and rich photophysical properties (Scheme 1)^[61-64]. The imine group (-C=N-) acts as the key functional group and plays multiple roles. It functions as an efficient fluorescent switch that enables signal variation via conformational transformation; it can coordinate with metal ions to construct stable metal-organic frameworks^[65,66]; and it is able to specifically interact with the amino and carboxyl groups of amino acids to achieve a synergistic chiral recognition effect, which facilitates the efficient identification of target molecules. More importantly, the precise introduction of chiral sources (e.g., chiral amines, chiral aldehydes) into the Schiff base scaffold enables the convenient construction of an intrinsic chiral microenvironment, which serves as the core structural basis for achieving enantioselective recognition of amino acid enantiomers^[67,68]. In recent years, probes based on Schiff bases have achieved fruitful results in fields such as metal ion and anion recognition^[69-72]. With the growing demand for chiral analysis, such probes have demonstrated significant application potential in the fluorescent detection of amino acid enantiomers, providing important support for the development of novel chiral sensing technologies.

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Scheme 1. Advantages of Schiff base fluorescent probes, types of chiral recognition substrates (amino acids), enantioselective recognition mechanisms, and application scenarios.

Based on the classification of amino acid types (including basic, acidic, non-polar, and polar), this paper summarizes the research progress of Schiff base fluorescent probes in the field of enantioselective recognition of amino acids over the past decade. It focuses on the discussion of probe molecular structure design, enantioselective spectral response behavior, molecular recognition mechanism, and their practical application value (Scheme 1). Finally, based on the key scientific challenges currently existing in this field, and combined with the structural characteristics and development trends of Schiff base compounds, the future research directions are prospected.

2. Enantioselective Recognition of Amino Acids by Schiff base Fluorescent Probes

The Schiff base fluorescent probes reviewed herein are capable of enantioselective recognition toward two categories of amino acids: free amino acids with both unprotected amino and carboxyl groups, as well as N-protected amino acids.

2.1 Enantioselective recognition of basic amino acids

Basic amino acids refer to those whose side chains contain basic functional groups. Under physiological pH conditions, these groups can be protonated and carry a net positive charge. The main basic amino acids include lysine (Lys), arginine (Arg), and histidine (His). In terms of physiological functions, apart from serving as core substrates for protein biosynthesis and supporting the construction and repair of tissue structures, these amino acids are also widely involved in various key metabolic processes. To date, most reported Schiff-base fluorescent probes for the chiral recognition of basic amino acids mainly target Arg, with the other two basic amino acids scarcely documented. L-Arg is not only a participant in the urea cycle, but also involved in the regulation of endothelial function, the control of vascular tone, and the synthesis of creatine and collagen. Meanwhile, it also plays an indispensable role in the immune response^[73]. In contrast, D-Arg exhibits unique physiological and pharmacological activities: it inhibits bacterial growth by competitively suppressing bacteria’s utilization of L-Arg, and also has a central excitatory effect and can prolong sleep duration^[74]. It can thus be seen that selective recognition of Arg enantiomers is of great significance for in-depth analysis of their specific action mechanisms in physiological processes.

In 2023, Guo et al.^[75] connected chiral BINOL units with rhodamine-based near-infrared (NIR) dyes through a condensation reaction, constructing a Schiff base (C=N)-structured NIR fluorescent probe (S)-1 (Figure 1a). With the synergistic effect of La³⁺ ions, this probe can achieve enantioselective recognition of L-Arg. Under an excitation wavelength of 690 nm, the system shows a significant fluorescence enhancement at 764 nm (Figure 1b). The results indicate that when the probe interacts with 18 pairs of amino acid enantiomers respectively in a 7.4 HEPES/MeCN = 49/1 system in the presence of 5 equivalents of La³⁺ ions, only L-Arg can induce obvious fluorescence enhancement, while D-Arg and other amino acids do not cause significant responses. The enantioselective fluorescence enhancement ratio ef [(ef = (I_L - I₀)/(I_D - I₀), I₀: Fluorescence intensity of the probe in the absence of amino acids] of the probe for L-/D-Arg reaches 52.5. Moreover, when interacting with Arg enantiomers, the system is also accompanied by rapid and visible color changes, facilitating naked-eye distinction (Figure 1c). Mechanism studies show that the imine group, as one of the key recognition sites in the probe, triggers the opening of the spirolactam ring through coordination with La³⁺ on the one hand, and forms specific interactions such as hydrogen bonds with the guanidyl group of Arg on the other hand. Correspondingly, the possible fluorescence turn-on photophysical mechanism underlying this process is as follows: before ring-opening, the lone-pair electrons on the amide nitrogen transfer to the excited fluorophore via the photo-induced electron transfer (PET) effect, resulting in fluorescence quenching; upon ring-opening, the lone-pair electrons of the amide nitrogen participate in a new p-π conjugated system, which greatly suppresses the PET process and thereby triggers fluorescence emission. Thereby, the probe realizes highly selective recognition accompanied by an obvious fluorescence response. Benefiting from its NIR emission property, the probe might exhibit deeper tissue penetration ability, lower photodamage, and weak background fluorescence interference in complex biological environments, showing good application potential in bioanalysis.

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Figure 1. (a) Molecular structure of Probe (S)-1 and its binding mode with Arg; (b) Fluorescence response toward 18 pairs of amino acids; (c) UV-vis absorption spectra of (S)-1 after interaction with La³⁺ and Arg enantiomers (inset: photographs of the corresponding solutions under room light). Reproduced with permission from reference^[75]. Copyright © 2023 John Wiley & Sons. UV-vis: ultraviolet-visible.

Subsequently, our group^[76] reported another chiral imine fluorescent probe (R)-2 and its isomer (R)-3 based on the BINOL backbone (Figure 2a). In the presence of zinc ions, these probes exhibit excellent chiral recognition ability for Arg in DMSO/H₂O = 29/1, with an ef [ef = (I_L - I₀)/(I_D - I₀), I₀: Fluorescence intensity of the probe] value of approximately 90. Studies have shown that probe (R)-2 exhibits a significant fluorescence enhancement at 438 nm, which is independent of the chirality of amino acids; this signal can be used for quantitative analysis of amino acid concentrations (Figure 2b). In contrast, at 513 nm, it shows a pronounced enantioselective fluorescence response, effectively distinguishing D-Arg from L-Arg (Figure 2c). Similar results were also observed for probe (R)-3. Therefore, these probes allow the simultaneous detection of both Arg concentration and enantiomeric composition. Under excitation at 363 nm, the fluorescence intensity of probe (R)-2 at 438 nm showed a good linear relationship with Arg concentration in the range of 0-30 mmol/L (R² = 0.994) (Figure 2d). Under excitation at 445 nm, the fluorescence intensity at 513 nm exhibited a cubic function with the ee value of D-Arg (R² = 0.9994) (Figure 2e). Utilizing these two fitted equations, the absolute errors for concentration determination of actual samples ranged from 0.09 to 1.44 mmol/L, and those for ee value determination were below 5%. These results demonstrate that the probe enables simultaneous and accurate determination of both the concentration and enantiomeric composition of Arg through dual-excitation-wavelength detection. Mechanistic investigations revealed that the 3’-quinoline imine groups in the probes engaged in amino acid molecular recognition via N-Zn(II) complexation, and this interaction synergistically enhanced the enantioselectivity of BINOL-aldehyde for amino acids. Density Functional Theory calculations confirmed that the complex formed by (R)-2 + Zn²⁺ with D-Arg exhibits higher thermodynamic stability than that formed with L-Arg (Figure 2a). This difference in stability is presumably responsible for the enantioselective fluorescence enhancement.

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Figure 2. (a) Molecular structures of probes (R)-2 and (R)-3, and the interaction mode between probe (R)-2 and enantiomers of Arg; (b) Fluorescence response of probe (R)-2 with D/L-Arg at an excitation wavelength of 363 nm and (c) 445 nm; (d) Fluorescence intensity at 438 nm versus the concentration of L-Arg (black line) and D-Arg (red line), the fitted linear relationship between the average value of I_438nm and concentration of Arg (blue) line; (e) Fluorescence intensity at 513 nm versus the ee values of D-Arg. Reproduced with permission from reference^[76]. Copyright © 2024 Science Press.

2.2 Enantioselective recognition of acidic amino acids

Acidic amino acids ionize to carry a negative charge under physiological pH. Among the common 19 chiral amino acids, the acidic ones include Asp and Glu. Of the two acidic amino acids, Glu is more widely investigated for chiral recognition by Schiff-base fluorescent probes, whereas studies on the chiral fluorescent recognition of Asp remain scarce. L-Glu is one of the core amino acids for protein composition and in vivo metabolism. As the most important excitatory neurotransmitter in the central nervous system, it is involved in important physiological processes such as synaptic transmission, learning, and memory, and also plays a pivotal role in amino acid metabolism and nitrogen balance^[77]. In contrast, D-Glu is not a component of proteins; it mainly exists in the cell walls of certain bacteria and specific bioactive peptides. Its physiological functions are related to the structural stability of microbial cells and some enzymatic reactions, and in recent years, it has also been found to potentially participate in mammalian neuromodulation^[78]. The two enantiomers exhibit significant chiral differences in biological distribution, metabolic pathways, and functions, reflecting the decisive influence of configuration on the biological roles of amino acids. Therefore, conducting research on the enantioselective recognition of Glu enantiomers is of great scientific significance for revealing the chiral configuration-dependent physiological functions and regulatory mechanisms of Glu at the molecular level.

In 2020, Zhao et al.^[79] designed and synthesized a DACH-BODIPY based fluorescent probe (1R,2S)-4 (Figure 3a). This probe innovatively combines pyridoxine-5’-phosphate (PLP) and chiral 1,2-diaminocyclohexane as recognition units, and achieves efficient enantioselective recognition of Glu through a dynamic imine exchange reaction. After reacting with D-Glu, the probe shows a significant fluorescence enhancement at 562 nm, which could be ascribed to the disappearance of the PET from the original imine group to the BODIPY fluorophore. In addition to exhibiting enantioselective fluorescence enhancement toward Glu, this probe also shows chemoselective fluorescence enhancement for Glu when compared with its response toward other common amino acids (Figure 3b,c). Mechanistic studies show that the imine group, as a key dynamic covalent site, achieves specific response to D-Glu through a dual mechanism: first, it undergoes a biomimetic transimination reaction with the amino acid, displacing and releasing the fluorophore to trigger the fluorescent signal; second, it cooperates with the adjacent phosphate group to construct a chiral microenvironment through non-covalent interactions such as hydrogen bonds, thereby realizing highly enantioselective recognition of D-Glu. This biomimetic transimination based on dynamic imine metathesis enables rapid exchange between chiral amine substrates and PLP-derived imine probes. Such strategies present notable advantages, including broad applicability toward diverse chiral primary amines and fast response kinetics facilitated by the dynamic imine metathesis pathway. Nevertheless, a critical limitation is that these systems typically require arylamines as auxiliary indicators to generate fluorescence signals. This probe not only provides a new method for the specific detection of chiral amino acids, but has also been successfully applied to the fluorescence imaging of D-Glu in living cells (Figure 3d), demonstrating its potential applications in in situ chiral analysis in biological systems and disease diagnosis.

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Figure 3. (a) Molecular structure of probe (1R,2S)-4 and fluorescence enhancement process upon binding with D-Glu; (b) Fluorescence response of probe (1R,2S)-4 toward 12 amino acids; (c) Kinetic curves of the fluorescence intensity (at 562 nm) of the probe (1R,2S)-4 over time upon reaction with D-Glu and L-Glu in aqueous solution; (d) Mouse mammary carcinoma cell (4T1 cells) imaging: Bright-field, Green field, Merge field of 4T1 cells incubated with probe (1R,2S)-4 (10 μM) (Top); Bright-field, Green field, Merge field of 4T1 cells incubated with probe (1R,2S)-4 (10 μM) + D-Glu (1 mM) (Bottom). Scale bar is 20 μm. Reproduced with permission from reference^[79]. Copyright © 2020 Elsevier.

In 2024, Zhang et al.^[80] developed a chiral fluorescent probe (R)-5, which features a BINOL core integrated with a Schiff base moiety (Figure 4a). In the presence of Zn²⁺, the probe exhibits high enantioselectivity, enabling the sensitive detection of D-Glu with a low limit of detection (LOD) of 70 nM. In addition to D-Glu (an acidic amino acid), this probe also exhibits an enantioselective fluorescence enhancement response toward D-Arg (a basic amino acid) at low concentrations (Figure 4b,c). Investigations into the intrinsic recognition mechanism have shown that the reversible cleavage and reformation of this C=N bond, assisted by Zn²⁺ coordination, enable selective binding to the target D-enantiomers. Furthermore, the probe exhibits a color change under ultraviolet (UV) light in solution, allowing for rapid visual screening. A paper-based sensor was fabricated to enhance practical applicability: the paper-based sensor prepared with (S)-5 showed a distinct color response to the L-amino acids prevalent in food samples (e.g., wheat, rice), while the (R)-5-based sensor enabled the visual detection of D-Arg with a low naked-eye detection limit of 0.13 mg/100 g, showcasing its potential for rapid chiral analysis in foodstuffs (Figure 4d). This work paves the way for the broader use of enantioselective fluorescent probes in areas such as food science and field-deployable diagnostic tools. However, the paper-based detection method developed therein still requires further investigation in terms of storage stability, reproducibility, and quantitative accuracy.

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Figure 4. (a) Molecular structure of probe (R)-5 and the interaction mechanism with chiral amino acids; (b) Fluorescence response of probe (R)-5 toward 20 amino acids; (c) Fluorescence bar graph of probe (R)-5 showing the fluorescence response of 20 amino acids at an emission wavelength of 540 nm; (d) Schematic illustration of sensor fabrication and its color change toward food samples under natural light and 365 nm UV light. Reproduced with permission from reference^[80]. Copyright © 2024 Elsevier.

2.3 Enantioselective recognition of non-polar amino acids

Non-polar amino acids have side chains mainly consisting of hydrophobic groups (e.g., alkyl groups), including Ala, valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), Trp, methionine (Met), and proline (Pro). In biological systems, they play key roles: their hydrophobic side chains drive protein folding to form a stable hydrophobic core to maintain the three-dimensional structure of proteins; they help form transmembrane regions of membrane proteins and act as “molecular spacers” to regulate protein stability, flexibility, and interactions with other biomacromolecules. Additionally, they participate in critical physiological processes like energy metabolism, signal transduction, and antioxidant defense. Analyzing their chirality is vital for studying their specific biological functions. Among these eight nonpolar amino acids, currently reported chiral recognition by Schiff-base fluorescent probes has covered Ala, Val, Met, Phe and Trp. The other three members (Leu, Ile, and Pro) are rarely involved. For Pro specifically, its α-NH₂ group and chiral carbon constitute a rigid pyrrolidine ring. The resulting steric hindrance hinders chiral probes from accessing its chiral center. Therefore, not only Schiff-base fluorescent probes but also probes with other structures can hardly achieve effective chiral recognition of Pro.

2.3.1 Enantioselective recognition of Ala

L-Ala, a common aliphatic non-polar protein-building amino acid, is widely involved in organismal energy metabolism, gluconeogenesis, and immune regulation, with a key role in muscle and liver metabolism^[81]. Its non-natural enantiomer D-Ala, not involved in protein synthesis, is critical for bacterial cell wall peptidoglycans (providing structural stability and drug resistance) and has recently been found to potentially regulate neural signals in some higher organisms^[82]. The two enantiomers show notable chiral differences in metabolism, structure-function, and biological distribution, highlighting configuration specificity’s role in life processes. Thus, researching their enantioselective recognition and function is vital for understanding amino acid chirality’s regulatory mechanisms in life and provides a scientific basis for developing new antibacterial targets, diagnostic tools, and chiral drugs.

In 2012, Hou et al.^[83] designed and synthesized a new type of chiral (S)-diamine fluorescent polymer probe (S)-6 (Figure 5a). This probe was prepared via a Schiff base polycondensation reaction between a benzaldehyde-containing unit and (S)-2,2’-binaphthyldiamine. It exhibits weak intrinsic fluorescence but shows a significant fluorescence “turn-on” response (probe transitions from a weakly fluorescent or non-fluorescent state to a highly fluorescent state) to Zn²⁺ (Figure 5a,b). The in-situ formed 1:1 zinc(II)-polymer complex enables highly enantioselective recognition of N-Boc-Ala, with an ef value of 6.9 [ef = (I_L - I₀)/(I_D - I₀), where I₀ is the fluorescence intensity of the probe], inducing a significant fluorescence enhancement for the L-enantiomer (Figure 5c). This can be attributed to the fact that when the proton of N-Boc-protected alanine interacts with the N atom in the Schiff base imine moiety of the chiral polymer backbone, the lone pair electrons on the N atom are inhibited from participating in the PET process, resulting in fluorescence enhancement. Mechanistic studies revealed that the imine group serves as the core recognition site: it coordinates with Zn²⁺ to construct a chiral cavity, and then undergoes specific interactions with N-Boc-Ala via nitrogen atoms, thereby driving enantioselective recognition and fluorescent signal output. Experiments showed that after adding the L-enantiomer, the probe solution emits bright blue fluorescence under UV light, which can be directly observed with the naked eye even at low concentrations, realizing simple, rapid, and visual detection of chiral molecules. This work investigated the highly enantioselective fluorescent recognition of N-Boc-Ala based on in-situ formed chiral polymer-metal complexes, providing a novel polymer material strategy for chiral sensing.

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Figure 5. (a) Molecular structure of (S)-6 and its binding mode with Zn(II); Recognition process to Boc-L-Ala; (b) Fluorescence response of (S)-6 toward different metal ions; (c) Fluorescence spectra of (S)-6 + Zn²⁺ in the presence of Boc-D-/L-Ala. Insert: Photographs showing fluorescence color changes under 365 nm UV light: (1) complex sensor only; (2) complex sensor + Boc-D-Ala; (3) complex sensor + Boc-L-Ala. Reproduced with permission from reference^[83]. Copyright © 2012 American Chemical Society.

In 2016, Munusamy et al.^[84] reported another BINOL-structured fluorescent probe (S)-7 bearing imine groups, which is capable of chiral recognition of Ala with the involvement of metal ions (Figure 6a). A structural characteristic of this probe is that both the 3- and 3’-positions of the BINOL scaffold are modified with imine groups. Different from the aforementioned (S)-6, which exhibits fluorescence enhancement upon interaction with metal ions, the fluorescence of this probe is quenched by Fe(III) (Figure 6b), owing to the suppression of excited-state intramolecular proton transfer of ligand (S)-7 upon addition of Fe(III). Subsequent interaction with the chiral isomers of Ala results in enantioselective fluorescence enhancement with an ef [ef = (I_L - I₀)/(I_D - I₀), I₀: Fluorescence intensity of the probe] value of 2.52 (Figure 6c). Studies on the recognition mechanism suggest that the imine group, serving as a key coordination site for Fe(III), participates in the formation of a chiral metal complex. During the recognition process, amino acids displace the original probe molecules through competitive coordination, thereby achieving the selective recognition and fluorescent response towards amino acid enantiomers (Figure 6a).

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Figure 6. (a) Molecular structure of probe (S)-7 and recognition process of Ala under the participation of Fe³⁺; (b) Bar graph of fluorescence response to different metals; (c) Fluorescence spectra in response to L-/D-Ala. Reproduced with permission from reference^[84]. Copyright © 2016 Elsevier.

Unlike the BINOL-based chiral fluorescent probe (S)-7 with a diimine structure, Han et al.^[85] designed and synthesized the BINOL-structured chiral fluorescent probe (R)-8 featuring a single imine group. In an acetonitrile system, the aldehyde group at the 3-position of this probe reacts with the amino group of amino acids to form an imine, and with the synergy of Zn²⁺, a stable ternary complex is generated, thereby triggering a significant fluorescence enhancement (Figure 7a). Probe (R)-8 exhibits extremely strong enantioselectivity towards D-Ala, with a LOD as low as 10.7 nM, and can specifically recognize D-Ala among 20 different amino acids (Figure 7b,c). Mechanistic studies revealed that the aldehyde group, acting as a key recognition site, reacts with D-Ala to form a C=N double bond. Subsequently, coordination occurs between Zn²⁺ and the nitrogen atom (from the C=N bond), carboxylate group, and hydroxyl group, leading to changes in the conjugated system and amplification of the fluorescence signal. Theoretical calculations and NMR results further verified the stereomatching of the recognition process and the stability of the complex. In addition, the probe has been successfully applied to test strip detection (completed within 3 minutes) and fluorescence imaging of D-Ala in HeLa cells (Figure 7d), demonstrating the potential for rapid recognition and visual detection of chiral amino acids in complex biological environments. However, cell-level chiral recognition experiments indicated that although D-Ala could induce significant fluorescence enhancement, L-Ala also caused noticeable signal increase, suggesting that the probe has only moderate chiral recognition specificity in cells. This limitation may mainly arise from the influence of complex matrices in cells. As observed from the solution fluorescence spectra, the emission wavelength of the probe toward amino acids is below 600 nm, falling within the short-wavelength region. In this range, interference from biological matrices is relatively severe, which causes significant disturbances to fluorescence detection. In addition, the complex intracellular environment may also affect the binding ability of the probe toward the target amino acid. However, the test strip detection results confirmed its simple and rapid qualitative/semi-quantitative analysis capability: under natural light, the test strip color changes from colorless to yellow with increasing D-Ala concentration; under 365 nm UV light, the fluorescence intensity gradually increases (Figure 7e), enabling instrument-free visual detection of D-Ala.

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Figure 7. (a) Molecular structure of probe (R)-8 and the interaction with chiral amino acids; (b) Fluorescence spectra and (c) bar chart of probe (R)-8 in the presence of 20 pairs of amino acids; (d) Confocal fluorescence microscopy images of HeLa cells: incubation with probe (R)-8 alone (A1-A3); incubation with probe (R)-8 + D-Ala + Zn²⁺ (D1-D3); incubation with probe (R)-8 + L-Ala + Zn²⁺ (E1-E3) and fluorescence intensity of probe (R)-8 for detecting chiral Ala in HeLa cells (right panel); (e) (R)-8 test strips after reaction with different concentrations of D-Ala under natural light and under 365 nm UV light illumination. Reproduced with permission from reference^[85]. Copyright © 2025 Elsevier. UV: ultraviolet.

2.3.2 Enantioselective recognition of Val

L-Val is a non-polar essential amino acid for humans, belonging to the category of branched-chain amino acids. It plays a crucial role in protein synthesis, regulation of muscle metabolism, maintenance of blood glucose stability, and tissue repair^[86]. In contrast, D-Val is an unnatural configuration that does not participate in mammalian protein synthesis. It mainly exists in certain antibiotics and bacterial peptidoglycans, and exhibits specific antibacterial biological activity^[87]. The two configurations show distinct chiral differences in metabolic pathways, biological functions, and distribution in biological systems. Therefore, the chiral recognition of Val holds significant theoretical research significance and practical application value for in-depth studies on its biological action mechanisms and functional differences.

In 2025, our research group^[88] designed and synthesized imine-type chiral fluorescent probes (S)/(R)-9 modified with 2-aminothioanisole, based on the chiral scaffold of BINOL (Figure 8a). A standout feature of these probes is their exceptional universality. As shown in Table 1, the probe exhibits high enantioselectivity toward 12 amino acids in EtOH/H₂O (1/1, v/v) and DMSO/H₂O (39/1, v/v) systems, respectively. Specifically, they exhibit high enantioselective fluorescence enhancement for Val in a DMSO/H₂O mixed solvent system at pH 7.5 (Figure 8b,c), with an ef [ef = (I_L - I₀)/(I_D - I₀), I₀: Fluorescence intensity of the probe] value exceeding 20. Remarkably, the probe achieved extremely high enantiomeric ef for various amino acids, such as 646.2 for asparagine (Asn) and 255.6 for Val in EtOH/H₂O, underscoring its powerful and versatile chiral discriminating capability. The recognition mechanism is based on a two-step “condensation-coordination” reaction: the aldehyde group of the probe condenses with the amino group of the amino acid to form an imine intermediate, which then forms a rigid “probe-amino acid-Zn²⁺” ternary complex under the synergistic effect of the 2-aminothioanisole group (Figure 8a). Fluorescent differentiation between D- and L-configured amino acids is achieved through conformational differences. The resulting rigid, chirally matched complex exhibits low non-radiative relaxation in the excited state, which may be one of the reasons for the significant fluorescence enhancement. Based on this recognition mechanism, it can be inferred that the broad-spectrum chiral recognition of amino acids by this probe may arise from the synergistic effect of the 2-aminothioanisole modification in facilitating the formation of rigid complexes during chiral recognition. We successfully applied this probe to realize chiral fluorescent imaging of Val in human normal liver cells (L-O2 cells) and zebrafish (Figure 8d,e). This study not only advances the practical application of chiral fluorescent probes in physiological environments but also provides important technical support for the diagnosis of diseases related to amino acid racemization, accurate analysis of amino acid configurations in biological systems, and in-depth research on disease mechanisms.

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Figure 8. (a) Molecular structure of probe (S)-9 and its recognition mechanism toward L-Val; (b) UV-Vis absorption spectra of probe (S)-9 + Zn²⁺ in the presence of D-/L-Val; (c) Enantioselective fluorescence response of probe (S)-9 + Zn²⁺ toward D/L-Val (Inset: photographs under 365 nm UV irradiation); (d) Fluorescence imaging of (S)-9 + Zn(II) + L-/D-Val in L-O2 cells and (e) zebrafish, scale bar in d is 40 μm, in e is 200 μm. Reproduced with permission from reference^[88]. Copyright © 2025 Elsevier. UV-vis: ultraviolet-visible.

2.3.3 Enantioselective recognition of Met

As a non-polar essential amino acid in humans, L-Met plays a crucial role in key physiological processes such as protein biosynthesis, methylation metabolism regulation, and antioxidant defense^[89]. In contrast, although D-Met does not directly participate in mammalian protein synthesis, it has demonstrated significant application potential in asymmetric synthesis and chiral drug preparation in recent years, serving as a chiral synthetic precursor and catalyst ligand^[90]. Currently, chiral conversion and enantiomeric recognition between L-Met and D-Met have become research hotspots; in-depth exploration of these processes not only helps improve the efficiency and selectivity of chiral synthesis but also provides new insights for the development of novel asymmetric catalytic systems.

In 2017, Ucar et al.^[91] successfully prepared a chiral Schiff base fluorescent probe (S)-10 based on ferrocene (Figure 9a). Spectroscopic studies showed that under excitation at 360 nm, the fluorescence intensity of probe (S)-10 increased significantly with the addition of D-Met. In contrast, L-Met and other studied chiral amino acids caused negligible fluorescence change, indicating that the probe possesses both enantioselective and chemoselective recognition ability for D-Met (Figure 9b,c). Further characterization revealed an ef [ef = (I_D - I₀)/(I_L - I₀), I₀: Fluorescence intensity of the probe] value of 1.54 for D-Met, along with a detection limit of 52 μM. Based on the ¹H NMR results and enantioselective fluorescence responses, it can be inferred that Met enantiomers may interact with the imine nitrogen atom, the substituted carbonyl oxygen atom of ferrocene, and the aryl ether oxygen atom of the probe molecule through hydrogen bonding interactions. Owing to the steric hindrance effect of the Met side chain, the D-enantiomer exhibits stronger intermolecular forces with the probe than the L-enantiomer. This study not only expands the application of ferrocene-derived Schiff base compounds in chiral fluorescent sensing but also provides new ideas for the development of novel D-Met-selective recognition materials, holding potential application value in the fields of chiral analysis and biosensing.

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Figure 9. (a) Molecular structure of probe (S)-10 and the interaciton with D-Met; (b) Fluorescence spectra of probe (S)-10 with 5 pairs of animo acids; (c) Fluorescence enhancement ratio of probe (S)-10 to 5 pairs of amino acids. Reproduced with permission from reference^[91]. Copyright © 2017 Springer.

In 2020, Zhao et al.^[54] reported a hydrophilic Schiff base-structured NIR fluorescent probe (R)-11 (Figure 10a). The sodium sulfonate moieties introduced at the 6- and 6’-positions of the chiral BINOL scaffold significantly enhanced the molecule’s water solubility. In the presence of Zn²⁺ and within a pH 7.4 HEPES/1% DMSO system, this probe exhibited NIR fluorescence enhancement responses at 730 nm toward 14 types of amino acids (including basic, polar, and non-polar amino acids). Among these, its chiral recognition effect for Met was particularly prominent. D-Met could induce a strong fluorescence enhancement, while the response to L-Met was weak, with an ef [ef = (I_D - I₀)/(I_L - I₀), I₀: Fluorescence intensity of the probe] value as high as 36.4 (Figure 10b). This remarkable difference enables the probe to be used for the accurate analysis of the enantiomeric composition of Met. The fluorescence response intensities of (R)-11 and (S)-11 showed a good mirror-image linear relationship with the optical composition of Met (Figure 10c). Mechanistic studies on chiral recognition revealed that, on one hand, the probe achieves specific binding through the condensation reaction between the aldehyde group at the 3-position and the amino group of amino acids; on the other hand, it regulates the activation of NIR fluorescence via the ring-opening reaction of its spiro-lactam unit, and cooperates with Zn²⁺ in synergistic coordination. These processes collectively enable highly enantioselective recognition of chiral amino acids (Figure 10a). This study represents the first achievement of highly enantioselective fluorescent recognition of Met in the NIR region, providing a novel and efficient tool for the imaging of chiral amino acids and the analysis of enantiomeric composition in biological systems.

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Figure 10. (a) Molecular structure of probe (R)-11 and its interaction mode with amino acids; (b) Fluorescence response of probe (R)-11 + Zn²⁺ toward D-/L-Met; (c) Relationship of fluorescence intensity of probe (R)-11 and (S)-11 with the enantiomeric purity of Met at an emission wavelength of 736 nm. Reproduced with permission from reference^[54]. Copyright © 2020 American Chemical Society.

In 2021, based on the NIR chiral fluorescent response of (R)-11, Chen et al.^[52] developed a pair of pseudoenantiomeric probes (a pair of structurally analogous probes with opposite configurations, which are not true enantiomers but exhibit enantiomer-like chiral recognition behavior, are defined as a pair of pseudoenantiomeric probes), (R)-13 and (S)-12, which exhibit complementary red and green fluorescence (Figure 11a). They proposed a novel dual-color fluorescent sensing strategy for the semi-quantitative visual analysis of chiral amino acids. This combination enables the simultaneous detection of the two enantiomers of chiral amino acids at two significantly different wavelengths (655 nm and 505 nm). For Met, the ef [ef = (I_D - I₀)/(I_L - I₀), I₀: Fluorescence intensity of the probe + Zn²⁺] value of this sensor is 2.5, further confirming its excellent enantioselectivity. Additionally, the sensor exhibits enantioselective recognition for a variety of other amino acids, including Leu, Lys, Trp, Phe, Arg, Val, Asp, His, and Glu, demonstrating good universality. When (R)-13 and (S)-12 were mixed in an equimolar ratio and reacted with Met samples of different ee values in the presence of Zn²⁺, the fluorescent color of the system showed a continuous change from green, yellow, orange to red under excitation of 365 nm as the ee value of D-Met gradually increased (Figure 11b,c). This phenomenon indicates that such pseudoenantiomeric probe combinations can achieve rapid, semi-quantitative visual analysis of the enantiomeric composition of chiral amino acids through intuitive fluorescent color changes. The study pointed out that the imine groups are involved in the formation of rigid dimeric complexes, which effectively inhibit non-radiative transitions, thereby realizing enantioselective fluorescence enhancement. This strategy represents the first achievement of visual semi-quantitative analysis of the enantiomeric composition of chiral compounds based on fluorescent color changes, providing a new idea for the development of chiral analysis technologies suitable for on-site rapid detection.

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Figure 11. (a) Molecular structure of probe (S)-12 and (R)-13; Emission wavelengths toward L/D-Met in the presence of Zn²⁺; (b) Fluorescence spectra of (S)-12 + (R)-13 + Zn²⁺ toward Met with different enantiomeric compositions; (c) Photographs under 365 nm illumination of probe (S)-12 + probe (R)-13 + Zn²⁺ with Met samples of different enantiomeric compositions. Reproduced with permission from reference^[52]. Copyright © 2021 American Chemical Society.

2.3.4 Enantioselective recognition of Phe

L-Phe is an essential amino acid in humans, playing a crucial role in protein synthesis, neurotransmitter metabolism, and acting as an allosteric modulator of the calcium-sensing receptor. It can regulate glucose homeostasis and hormone secretion^[92]. In contrast, D-Phe does not participate in mammalian protein synthesis but possesses pharmacological potential such as inhibiting the activity of certain enzymes and exerting analgesic effects. In recent years, it has also attracted attention in the research of chiral drug synthesis and neuromodulation^[93]. Enantiomers of Phe exhibit significant chiral differences in metabolic pathways, receptor interactions, and biological functions. Conducting chiral recognition studies on them is of great significance for understanding the stereospecific functions of amino acids and developing targeted drugs.

In 2015, Wen et al.^[51] reported a chiral fluorescent probe (R)-14 with a bisimine structure based on a BINOL scaffold (Figure 12a). This probe can generate differential responses toward chiral functional amines including Phe at two emission wavelengths. At λ₁ = 427 nm, it exhibits a concentration-dependent but chirality-independent fluorescence enhancement (denoted as I₄₂₇), which is suitable for the determination of substrate concentration. At λ₂ > 500 nm, it shows a highly enantioselective fluorescence enhancement (denoted as I₅₀₉) (Figure 12b,c,d), enabling the analysis of enantiomeric composition. Figure 12e presents a three-dimensional plot of I₄₂₇, I₅₀₉, and L-Phe%. This plot can be used to determine the enantiomeric composition of Phe with varying concentration. Mechanistic studies revealed that I₄₂₇ originates from the substitution of the 2-naphthylamine unit by amino acids and the subsequent fluorescence recovery of 2-naphthylamine, while I₅₀₉ arises from the stereoselective binding between the chiral binaphthyl unit and the substrate, as well as the complexation with Zn(II) (Figure 12a). The imine groups play a core role in the recognition process: on the one hand, they undergo a displacement reaction with the amino groups of amino acids to form dynamic covalent bonds, achieving specific recognition and binding of the substrate; on the other hand, they form a rigid-structured complex with the synergy of Zn²⁺, which in turn triggers the activation of fluorescence signals and ultimately enables efficient discrimination of amino acid enantiomers. This strategy provides a new insight for the development of universal chiral sensing platforms and is expected to be widely applied in the rapid analysis of diverse chiral substrates.

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Figure 12. (a) Structure of probe (R)-14 and proposed reaction mechanism with chiral amines; (b) Fluorescence spectra of probe (R)-14 toward different concentrations of L-Phe and (c) D-Phe; (d) Fluorescence intensity at 509 nm of probe (R)-14 versus D/L-Phe concentration; (e) Fluorescent responses of I₄₂₇ and I₅₀₉ for the interaction of (R)-14 with Phe of varying concentrations and enantiomeric compositions. Reproduced with permission from reference^[51]. Copyright © 2015 American Chemical Society.

In 2021, Zhao et al.^[94] developed a chiral fluorescent sensor 15 (Figure 13) based on a carbazole-conjugated COF for the high-sensitivity fluorescent recognition of Phe enantiomers. This material was synthesized via a one-pot strategy: first, the chiral monomer CTp was prepared by the reaction of chiral (+)-diacetyl-L-tartaric anhydride ((+)-Ac-L-Ta) with 1,3,5-triformylphloroglucinol; subsequently, the chiral fluorescent sensor with optical activity was constructed through a Schiff base condensation reaction between CTp and 3,6-diaminocarbazole (Figure 13). The resulting material exhibits a high specific surface area (88.4 m²/g), tunable pore size (1.07-2.12 nm), good thermal stability (thermal decomposition temperature > 310 °C), and a significant chiral Cotton effect. As can be seen from the fluorescence responses shown in Figure 13, the synthesized COF probe exhibits a certain chiral recognition effect toward Phe, with an ef value (I_D/I_L) of approximately 1.6. Within the concentration range of 0.05-5 μmol/L, the material displays a good linear relationship with D-Phe and L-Phe, with LOD reaching 0.027 μmol/L and 0.037 μmol/L, respectively. In the spiked recovery experiment of actual samples, the recovery rate ranges from 84.8% to 99.0%, indicating that this method has good applicability and reliability in complex matrices. Furthermore, recycling experiments reveal that using methanol/water (1:1, v/v) as the eluent, Phe enantiomers can be completely desorbed from the porous framework, and the probe maintains stable chiral recognition efficiency and fluorescence response without obvious performance attenuation after five consecutive adsorption–elution cycles. The chiral recognition mechanism mainly relies on the non-covalent interactions including hydrogen bonding, electron transfer, and π–π stacking of imine groups, which cooperate with chiral centers to achieve selective recognition of chiral amino acids. The fluorescence enhancement mechanism can be explained as follows: the carbazole electron donor and methoxy electron acceptor endow the probe with intramolecular PET, leading to fluorescence quenching. The non-covalent interaction between Phe and the probe weakens this PET effect, thereby resulting in fluorescence enhancement. Compared with homogeneous probes, COF-based probes exhibit significant advantages in recyclability and stability. However, the enantioselectivity of current chiral COF probes remains moderate. This may be attributed to the fact that the enantioselective recognition mechanism of chiral COF probes mainly relies on noncovalent interactions, and the introduced chiral microenvironment is strongly influenced by the COF framework, which may lead to inferior stereoselectivity compared with the same chiral moiety in a homogeneous system. How to integrate the superior stability and recyclability of COF materials with the high enantioselectivity of homogeneous probes will be a key research direction in this field.

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Figure 13. Synthesis route of probe 15 and its fluorescence response to L-/D-Phe. Reproduced with permission from reference^[94]. Copyright © 2021 Elsevier.

2.3.5 Enantioselective recognition of Trp

L-Trp is an essential amino acid for humans. As a precursor of neurotransmitters and hormones such as serotonin and melatonin, it plays a crucial role in regulating mood, sleep, and neurological functions, and is also involved in protein synthesis and immune regulation^[95]. In contrast, D-Trp does not participate in protein synthesis in mammals, but can serve as a component of certain antibiotics and bioactive peptides. Recent studies have shown that it may affect microbial metabolism and possesses neuroregulatory potential^[96]. These two enantiomers exhibit significant chiral differences in metabolic pathways, physiological functions, and biological distribution. Their recognition and research are of great significance for understanding nervous system functions, developing neuropharmaceuticals, and exploring novel antibacterial strategies.

In 2017, Wolf et al.^[97] developed a biomimetic indicator displacement assay based on PLP (Figure 14a), utilizing an imine displacement mechanism similar to that of (R)-14 (Figure 12a). This assay enables the simultaneous and rapid determination of the absolute configuration, enantiomeric composition, and total concentration of unprotected amino acids, amino alcohols, and other functional chiral amines. Leveraging the reaction mechanism where PLP forms Schiff bases with amino groups, the researchers designed and synthesized PLP aromatic imine probes (e.g., probes 16-18, Figure 14b), which covalently capture chiral substrates containing primary amino groups through a highly favorable and quantitative imine metathesis reaction. This strategy relies on dual signal outputs of circular dichroism (CD) and fluorescence: the chiral PLP-imine complexes induce a characteristic Cotton effect (Figure 14c), which is used to identify the absolute configuration and calculate the ee value; meanwhile, the displaced aromatic amine indicator produces an “off-on” fluorescence response independent of enantiomeric composition, enabling accurate quantification of the total concentration. The reaction in this system can be completed within minutes in methanol, and it exhibits excellent versatility for 19 chiral compounds (including various amino acids, amines, and amino alcohols). Taking the detection of Trp using Probe 18 as an example, the CD response shows a linear correlation with the ee value (Figure 14d), and the determination error falls within the range allowed for high-throughput screening (typically ≤ 10% ee). This method eliminates the need for sample pre-derivatization, features simple operation and low cost, and provides a successful example of the first application of PLP in chiral sensing. It also offers a new approach for the development of efficient high-throughput chiral analysis platforms.

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Figure 14. (a) Concept of the PLP chiral sensor; (b) Molecular structures of probes 16-18; (c) CD spectra of probe 16 toward L-/D-Trp; (d) Linear relationship between the CD signal of probe 18 and the ee value (%) of Trp. Reproduced with permission from reference^[97]. Copyright © 2017 American Chemical Society. CD: circular dichroism.

2.4 Enantioselective recognition of polar amino acids

Polar amino acids whose side chains contain polar functional groups (e.g., -OH, -SH, or -CONH₂) carry no net charge under neutral pH conditions. This category of chiral amino acids mainly includes Ser, threonine (Thr), cysteine (Cys), Asn, Gln, and tyrosine (Tyr). Their side chains exhibit strong hydrophilicity and can form specific interactions with water molecules or other biological macromolecules through hydrogen bonding. Consequently, they play crucial roles in processes such as maintaining the three-dimensional structure of proteins, mediating molecular recognition, and acting as key residues in enzyme active sites to participate in catalytic reactions. At present, research on imine-based fluorescent probes for chiral recognition of such polar amino acids remains very limited. Accordingly, we only discuss the reported imine probes for chiral recognition of Thr and Tyr in this section.

2.4.1 Enantioselective recognition of Thr

As a polar essential amino acid for humans, L-Thr is a key component in protein synthesis and immune regulation, and is widely involved in maintaining cellular metabolism and physiological functions^[98]. In contrast, its stereoisomer D-Thr does not participate in protein synthesis in mammals. However, studies have shown that D-Thr can affect the formation and stability of biofilms in bacteria. For instance, D-Thr inhibits biofilm formation of certain pathogenic bacteria by interfering with the assembly of extracellular matrices, and may act as a substrate or inhibitor in some enzymatic reactions to participate in the regulation of microbial metabolism^[99]. Due to differences in their spatial configurations, these two enantiomers exhibit fundamental differences in biological activity, metabolic pathways, and functional mechanisms. Therefore, the accurate recognition and differentiation of L-Thr and D-Thr not only hold crucial theoretical significance for studying the biological functions of amino acids, but also provide an important basis for developing novel anti-biofilm drugs, exploring microbial group behavior, and designing chiral-specific molecular tools.

In 2019, Iqbal et al.^[100] designed and synthesized a compound (R)-19 based on a tetrabrominated BINOL structure. This compound undergoes a condensation reaction with tetrabutylammonium (TBA) salt of L-Trp to form a Schiff base-type probe (R)-20 (Figure 15a). Subsequently, the authors investigated the fluorescence response of this probe toward the TBA salts of various amino acid enantiomers. In a HEPES/1% DMSO mixed system at pH 7.4 and in the presence of Zn²⁺, probe (R)-20 exhibits fluorescence emission at 350 nm that is independent of Thr chirality and only correlated with its concentration; in contrast, it shows a significant enantioselective fluorescence enhancement towards Thr at 500 nm (Figure 15b) with an ef [ef = (I_D - I₀)/(I_L - I₀), I₀: fluorescence intensity of the probe + Zn²⁺] value of 93.2. Moreover, the probe also demonstrated recognition capability for TBA salts of other amino acids including Leu, Met, Tyr, Val, Phe, and Glu, all of which exhibited similar dual-emission responses, enabling simultaneous determination of both concentration and enantiomeric composition. Probe (R)-20 provides an efficient and convenient dual-mode detection strategy for the simultaneous acquisition of Thr concentration and stereoconfiguration information. The dual-mode detection mechanism of probe (R)-20 shares similarities with that of the aforementioned probe (R)-14 (Figure 12a): in both cases, the imine group (C=N) in the probe acts as a dynamic covalent bond, enabling specific capture of the substrate through a reversible imine exchange reaction with the amino group of the amino acid. The difference, however, lies in the short-wavelength emitters released from the two different chromophores, which are L-Trp and 2-naphthylamine, respectively.

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Figure 15. (a) Molecular structure of the compound (R)-19 and synthetic route of probe (R)-20; Chiral recognition process to Trp; (b) Fluorescence response of probe (R)-20 toward Thr. Reproduced with permission from reference^[100]. Copyright © 2019 John Wiley & Sons.

2.4.2 Enantioselective recognition of Tyr

L-Tyr is a common polar amino acid in protein composition and also a precursor of key neurotransmitters and hormones such as dopamine and adrenaline. It is crucial for maintaining nervous system function and metabolic balance^[101]. In contrast, D-Tyr exerts potential effects in inhibiting bacterial biofilm formation and regulating enzyme activity^[102]. The two enantiomers of Tyr exhibit significantly different metabolic pathways and physiological functions in organisms. The chiral analysis of Tyr not only helps reveal the basic laws of chiral recognition in biological systems, but also provides new perspectives and tool foundations for drug development, microbial behavior regulation, and neuroscience research.

In 2024, Yuan et al.^[103] developed a COF fluorescent sensor, probe 21 (Figure 16a). This material was synthesized from the monomers 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 2,5-dimethoxyterephthalaldehyde (DMTP), and subsequently modified via a chiral monomer exchange strategy using helicid (HD). The resulting COF maintained high crystallinity and excellent porosity with a BET specific surface area of 2338 m²/g, while inheriting strong fluorescence from the parent framework. More importantly, the incorporated chiral hydroxyl groups from HD, together with the imine bonds in the framework, created a chiral microenvironment, enabling a preferential fluorescence enhancement response towards L-amino acids such as L-Tyr (Figure 16b,c), with an enantiomeric fluorescence enhancement factor (ef = K_L/K_D, derived from Benesi–Hildebrand analysis) being below 2, indicating moderate chiral discrimination. As shown in Figure 16d,e, both L-Tyr and D-Tyr can form hydrogen bond interactions with the hydroxyl groups of chiral HD in HD-TAPB-DMTP COF. The formation of host-guest complexes between the probe and amino acids may restrict non-radiative rotational relaxation of the HD-TAPB-DMTP COF and affect the π-π interactions between its layers, thereby leading to enhanced fluorescence intensity. This study not only expands the synthesis strategy for defective chiral COFs but also provides a new approach for the application of such materials in the fields of biomolecular recognition and chiral sensing, fully demonstrating the potential of chiral COFs in the enantioselective recognition of amino acid enantiomers. Of course, this probe still has some limitations in terms of chirality selectivity, which might arise from several factors: a low incorporation ratio of the chiral monomer HD (HD:DMTP ≈ 1:22), resulting in insufficient chiral recognition sites; marginal differences in hydrogen bonding and binding energy between enantiomers, as revealed by molecular docking; a large pore size (~3.3 nm) that provides weak steric confinement; and a fluorescence response that relies solely on interlayer π–π interactions, which cannot amplify subtle binding differences into a selective signal. To address these limitations, rational design strategies for COF-based sensors include increasing chiral site density via multidentate chiral monomers or post-synthetic modification, optimizing pore architecture to strengthen steric confinement, introducing multiple noncovalent interactions to amplify enantiomeric binding energy differences, integrating AIE or donor–acceptor units to enhance fluorescence sensitivity.

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Figure 16. (a) Molecular structure of probe 21; (b) Fluorescence spectra of probe 21 with different concentrations of L-Tyr and (c) D-Tyr; (d) Schematic diagram of the interaction between probe 21 and L-Tyr, (e) D-Tyr. Reproduced with permission from reference^[103]. Copyright © 2024 John Wiley & Sons.

3. Summary and Outlook

3.1 Summary of current research

Over the past decade, Schiff base fluorescent probes have evolved into powerful tools for the enantioselective recognition of amino acids, benefiting from their simple synthesis, modifiable structures, and superior coordination properties. Substantial advances have been accomplished in probe design, recognition mechanism elucidation, and practical application exploration. With respect to target analytes, enantioselective discrimination has been successfully realized for a diverse range of amino acid species. As summarized in Table 2, research on acidic amino acids has predominantly focused on Glu, whereas investigations into basic amino acids have centered on Arg. Non-polar amino acids represent the most widely investigated class, covering Ala, Val, Met, Phe, and Trp. By comparison, research into polar amino acids has been limited to Thr and Tyr, leaving critical gaps in the recognition systems for Ser, Asn, and Gln. In terms of recognition of performance and mechanisms, these probes generally exhibit fluorescence enhancement responses. Some systems achieve “off-on” or dual-color fluorescence switching and possess high sensitivity. For instance, probe (R)-5 has a detection limit of 70 nM for D-Glu, and probe (R)-8 for D-Ala reaches an exceptionally low LOD of 10.7 nM, meeting the requirements for trace biological sample detection. As also illustrated in Table 2, recognition systems are predominantly based on aqueous-organic mixed solvents (e.g., DMSO/H₂O, HEPES/MeCN), with pH conditions typically within the physiologically relevant range (7.0-7.5). Only small-molecule probe 4 modified with hydrophilic phosphate groups and COF probe 21 functionalized with polar chiral hydroxyl groups are capable of chiral recognition of amino acids in water. The reasons include: the poor water solubility of the probes; the strong affinity of water for amino acids and the hydrogen-bonding interactions significantly interfere with the binding between probe molecules and amino acids; the solvation effect of water and the aggregation of organic fluorescent molecules in aqueous media usually exert considerable influence on the fluorescence emission of probes. It is thus evident that the construction of highly sensitive chiral fluorescent sensors for amino acids in aqueous media at physiological pH remains a challenging task. Especially for chiral analysis in biological systems where aqueous environments are unavoidable, probes with aqueous-phase chiral recognition capability are particularly indispensable. In terms of recognition mechanisms, as shown in Table 2, they primarily rely on the synergistic coordination of the C=N groups of Schiff bases and the amino or carbonyl groups of amino acids with metal ions (e.g., Zn²⁺, La³⁺, Fe³⁺), while generating an enantioselective response in the chiral microenvironment of the probes. Additionally, mechanisms such as hydrogen bonding, imine exchange, π-π stacking, and spirolactam ring-opening are also widely involved in the recognition process.

In terms of the substrate coverage of chiral recognition, numerous reported Schiff base probes possess broad substrate applicability. For example, probes (S)-9 and (R)-9, functionalized with 2-aminothioanisole on a BINOL scaffold, exhibit exceptional universality. They can enantioselectively recognize a wide range of ten amino acids, including Asn, Glu, Val, Leu, Met, Ser, Gln, Arg, Phe, and Ala, in solutions at physiological pH, showcasing excellent adaptability and versatile chiral discriminating capability. Similarly, the pseudoenantiomeric probe pair comprising (R)-12 and (S)-13 enables dual-color fluorescent sensing and visual semi-quantitative analysis of the enantiomeric composition for multiple amino acids, including Met, Leu, Lys, Trp, Phe, Arg, Val, Asp, His, and Glu, demonstrating good generality. Furthermore, the PLP-based chiral sensor (Probes 16-18) also exhibits excellent versatility for 19 chiral compounds, including various amino acids, amines, and amino alcohols, achieving simultaneous determination of absolute configuration, enantiomeric composition, and total concentration. In the field of application development, certain Schiff base probes have exhibited substantial practical potential. For instance, COF-based probe 15 achieves highly sensitive recognition of Phe enantiomers in pure aqueous systems, with spiked recovery rates of 84.8%-99.0%, making it suitable for food safety monitoring. Furthermore, probes with low toxicity and good biocompatibility (e.g., (S)-9 for Val, (R)-8 for Ala) have been successfully applied for in situ fluorescence imaging in HeLa cells, L-O2 cells, and zebrafish models, providing visualization tools for studying diseases related to amino acid racemization.

3.2 Research prospects

Although notable advances have been achieved in this field, ample room for optimization remains for Schiff base fluorescent probes dedicated to the enantioselective recognition of amino acids. Hereafter, the future research directions and prospects of Schiff-base fluorescent probes for chiral recognition of amino acids will be discussed from three aspects: expansion of substrate scope, specific chiral recognition, elucidation of chiral recognition mechanisms, and practical applications.

3.2.1 Expansion of substrate scope

As mentioned above, among the chiral Schiff base probes reported to date, some common amino acids remain rarely explored as target analytes. In particular, polar amino acids, which constitute a major category, have received only limited investigation. Thus, developing rational strategies to realize efficient enantioselective recognition of polar amino acids using chiral Schiff base probes represents a key research direction for future work. Due to the structural characteristics of their polar side chains, such amino acids possess multiple interaction sites including hydrogen bonding, electrostatic, and dipolar sites, making it difficult to regulate probe chiral selectivity toward them. Moreover, water molecules in aqueous solution compete strongly for hydrogen bonding with these amino acids, which weakens the binding interaction and selectivity of probes toward them. Consequently, achieving highly enantioselective recognition remains challenging not only for imine-based probes but also for other types of probes. Therefore, molecular design strategies for high chiral selectivity targeting the structural features of their polar side chains are particularly crucial. Such as Ser, a hydroxyl-containing polar amino acid, it is possible to introduce strong Lewis acid groups, such as boronic acid groups, onto the chiral framework of the imine-type probe. By utilizing the coordination interaction between B and the imine N atom, as well as the covalent interaction with the hydroxyl of the Ser side chain, these factors can synergistically enhance the probe’s chiral recognition ability for Ser. Additionally, it is also possible to introduce hydrophilic groups onto the chiral framework to overcome the hydrogen bond competition in the aqueous phase, thereby increasing the interaction force between the probe and Ser and enhancing the chiral fluorescence response. And for Asn and Gln, two representative polar amino acids with amide groups, pyridine or imidazole moieties can be introduced to interact with the amide groups of target amino acids via hydrogen bonding; when these functional groups are integrated with the chiral scaffold, they contribute to the construction of stereoselective chiral cavities, thereby could effectively discriminate between the D- and L-enantiomers.

Beyond these polar amino acids with neutral polar side chains, Cys stands out as a uniquely challenging yet biologically significant target owing to its distinctive reactive thiol functional group, which endows it with completely different binding characteristics compared to hydroxyl- and amide-containing polar amino acids. For Cys, the incorporation of maleimide, α,β-unsaturated ketone, or haloacetyl moieties into chiral Schiff base probe molecules may facilitate the cooperative dual-site recognition of the thiol and amino groups within Cys. These thiol-specific reactive units may synergistically interact with the C=N motif of the Schiff base, triggering pronounced fluorescence “turn-on” or ratiometric responses. Such targeted structural engineering will endow the probes with high enantioselectivity and strong anti-interference capability, thereby demonstrating great potential for the precise enantioselective recognition of Cys in complex biological matrices.

In addition, we found that although nonpolar amino acids are the most extensively investigated targets in chiral recognition studies of Schiff base-type fluorescent probes, Pro, a nonpolar amino acid containing a pyrrolidine ring, has scarcely been mentioned. Pro differs structurally from other linear-chain nonpolar amino acids; in particular, its sterically hindered imine group renders specific recognition highly challenging. The incorporation of flexible alkyl or cycloalkyl moieties into chiral Schiff base probes may facilitate the stereospecific recognition of Pro via interactions with its sterically hindered imine group. These spatially matched hydrophobic moieties can synergistically interact with the C=N motif of the Schiff base via hydrogen bonding and hydrophobic effects, which effectively alleviates the steric hindrance induced by the pyrrolidine ring and reinforces chiral recognition interactions.

3.2.2 Specific chiral recognition

Furthermore, though substrate-universal chiral fluorescent probes with enantioselective recognition capability for multiple amino acids offer considerable advantages in application scenarios involving a single type of amino acid, specific enantioselective recognition of a particular amino acid becomes especially vital in environments containing multiple amino acid types, such as biological systems. How to construct a specific enantioselective recognition probe system based on the unique structural characteristics of amino acids remains a major challenge in current research within this field. For instance, Arg could be targeted via multiple hydrogen bonds and metal ion coordination towards its guanidino group; His might utilize metal ion coordination with the imidazole ring to build a ternary recognition system; and Phe selectivity could be enhanced by relying on π-π stacking and chiral cavities, thereby effectively avoiding cross-responses.

3.2.3 Elucidation of chiral recognition mechanism

The main structures of chiral α-amino acids are very similar, differing only in their side-chain groups. Although the molecular design of fluorescent probes based on the different interaction sites provided by side-chain groups is an important strategy to achieve the molecular recognition of target amino acids, the interaction mechanisms involved in chiral recognition are complex. In addition to the interactions between the probe and the side-chain groups of amino acids, the stereochemical matching between the probe and amino acids, including spatial complementarity, hydrogen bonding orientation, hydrophobic/hydrophilic interactions, and π-π stacking, as well as the microenvironment of the recognition system (e.g., solvent polarity and pH) can all affect the chiral recognition process. Therefore, the elucidation of the chiral recognition mechanism is not only of great guiding significance for the design of imine-based probes but also for the design of other types of chiral probes.

At present, the chiral recognition mechanisms of amino acids by most probes are mainly inferred from experimental phenomena and molecular docking simulations, while direct experimental evidence, especially from single-crystal X-ray diffraction of probe-amino acid complexes, is relatively limited. This lack of direct structural information hinders a comprehensive understanding of how probes differentiate between D- and L-amino acids at the molecular level.

There is still much research to be carried out in the future for the elucidation of the stereochemical basis of chiral recognition, including preparing single crystals of probe-amino acid complexes to directly observe the spatial interaction modes between the probe and D-/L-amino acids; combining in situ spectral techniques with quantum chemical calculations to analyze the dynamic changes of interaction sites during the recognition process; and designing structure-activity relationship studies by modifying the functional groups of chiral probes to clarify the key structural factors that affect stereoselective recognition.

3.2.4 Practical applications

For chiral recognition of amino acids in real environments, a critical issue to be addressed is the anti-interference capability of the probe. When employed in actual biological systems, the autofluorescence of organisms, biological matrices, trace metal ions, and other species constitute interfering factors that affect the chiral recognition of target amino acids. Most biological substrates (such as cells, tissues, and bodily fluids) produce inherent autofluorescence mainly in the short-wavelength region. A simple imine condensation enables the grafting of near-infrared chromophores onto chiral scaffolds, affording probe systems with long-wavelength emission and a large Stokes shift, which can effectively avoid spectral overlap with the autofluorescence of biological samples, thereby significantly suppressing background interference. Also, complex biological matrices contain various biomolecules (proteins, nucleic acids, amino acids, etc.) and metal ions that may interfere with fluorescence signals. Accordingly, the following strategies can serve as effective anti-interference approaches: introducing specific recognition moieties to improve selectivity; using fluorescence lifetime imaging to distinguish specific signals from matrix noise; employing chelation adjustment to optimize the coordination structure of probes, thereby enhancing their binding selectivity and stability toward target metal ions and reducing interference from competing metal ions; and applying appropriate sample pretreatment methods to alleviate matrix effects.

Moreover, the chiral recognition of amino acids by fluorophilic probes in the hydrophobic and lipophobic fluorinated phase can effectively eliminate interference from lipophilic and water-soluble impurities. This strategy can greatly improve the anti-interference performance of chiral fluorescent probes when applied to high-throughput chiral screening in asymmetric synthesis. Moreover, incorporating novel materials such as COFs and MOFs can boost the chiral recognition performance of the imine-based probes and extend their application scenarios.

In addition to chiral recognition toward single targets, real detection scenarios often require simultaneous analysis of multiple coexisting analytes.

For small-molecule imine-based probes, taking advantage of their structural tunability, distinct specific recognition functional groups can be introduced for different analytes respectively. Alternatively, molecular fragments containing different specific recognition moieties can be directly linked via imine condensation reactions to construct multifunctional probe architectures, thereby enabling simultaneous recognition of multiple analytes. This is expected to become a challenging research direction for chiral fluorescent probes in the future.

Furthermore, probe arrays have emerged as a promising strategy for simultaneous recognition of multiple analytes in complex mixtures via pattern-recognition-based response signals. Introducing the advantages of chiral imine-type probes into probe arrays enables simultaneous chiral analysis of multiple analytes in complex systems. Future efforts will concentrate on the rational design of chiral probe arrays with tailored binding affinity and signal output modes, with the goal of achieving simultaneous high-throughput chiral analysis of multiple amino acids in real samples.

To conclude, Schiff base fluorescent probes represent promising candidates for the enantioselective recognition of amino acids. By virtue of meticulous molecular engineering, breakthroughs in recognition mechanisms, and iterative optimization of practical applications, these probes will offer powerful analytical tools to propel developments across life sciences, medical diagnosis, and food safety monitoring.

Authors contribution

Wei J: Writing-original draft, methodology.

Li Y: Methodology, writing reviewing & editing.

Zhu Y, Gu S: Conceptualization, supervision, funding acquisition, writing-review & editing.

Conflicts of interest

The authors declare no conflicts of interest.

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

Not applicable.

Funding

The work was supported by the National Natural Science Foundation of China (Grant Nos. 22074114, 22377097, and 22307036), and Graduate Education Innovation Fund of Wuhan Institute of Technology (Grant No. CX2024030).

Copyright

© The Author(s) 2026.