Enantioselective Fluorescent Recognition of Free Amino Acids: Challenges and Opportunities

Fluorescent probes that can discriminate enantiomers of amino acids in organic media or aqueous solution are discussed. This Minireview focuses on recent progress in the studies of three classes of probes including those made of cyclodextrins, 1,1′-binaphthyl compounds, and nanomaterials, and uses them to illustrate the design strategies, applications, and limitations in this area. These probes are potentially useful for rapid analysis of asymmetric reactions for amino acid synthesis as well as the real-time imaging of amino acids in biological systems. The challenges in these applications are analyzed. Working in this field of enantioselective fluorescent recognition of amino acids offers great opportunities to make new scientific discoveries and to develop important practical applications.     

Simultaneous Determination of Concentration and Enantiomeric Composition of Amino Acids in Aqueous Solution by Using a Tetrabromobinaphthyl Dialdehyde Probe

3,3′-Diformyl-1,1′-bi-2-naphthol or its methoxymethyl-protected derivative is found to undergo a highly selective reaction with excess bromine in CH₂Cl₂ at reflux to give the novel 5,5′,6,6′-tetrabrominated product (S)- or (R)- 2 . The observed electrophilic substitution at the 5,5′-positons of an optically active binaphthyl compound is unprecedented. Unlike unbrominated 3,3′-diformyl-1,1′-bi-2-naphthol, which is not suitable for fluorescent recognition in water, compound (S)- 2 , in combination with Zn²⁺, exhibits a highly enantioselective fluorescent response toward amino acids in aqueous solution (HEPES buffer, pH 7.4). It is further found that the condensation product of (R)- 2 with tryptophan, (R)- 3 , shows dual-responsive emissions toward amino acids; the short wavelength (λ₁=350 nm) emission is sensitive to the concentration of the substrate regardless of the chiral configuration and the long wavelength (λ₂>500 nm) emission is highly enantioselective. Thus, the use of (R)- 3 allows the simultaneous determination of the concentration and enantiomeric composition of an amino acid sample from one fluorescence measurement.     

Construction of a Selective Fluorescent Probe for GSH Based on a Chloro‐Functionalized Coumarin‐enone Dye Platform

Glutathione (GSH), the most abundant intracellular biothiol, protects cellular components from damage caused by free radicals and reactive oxygen species (ROS), and plays a crucial role in human pathologies. A fluorescent probe that can selectively sense intracellular GSH would be very valuable for understanding of its biological functions and mechanisms of diseases. In this work, a 3,4‐dimethoxythiophenol‐substituted coumarin‐enone was exploited as a reaction‐type fluorescent probe for GSH based on a chloro‐functionalized coumarin‐enone platform. In the probe, the 3,4‐dimethoxythiophenol group functions not only as a fluorescence quencher through photoinduced electron transfer (PET) to ensure a low background fluorescence, but also as a reactive site for biothiols. The probe displays a dramatic fluorescence turn‐on response toward GSH with the long‐wavelength emission (600 nm) and significant Stokes shift (100 nm). The selectivity of the probe toward GSH over cysteine (Cys), homocysteine (Hcy), and other amino acids was demonstrated. Assisted by laser‐scanning confocal microscopy, we have demonstrated that the probe could specifically sense GSH over Cys/Hcy in human renal cell carcinoma SiHa cells.     

Highly Enantioselective Extraction of Underivatized Amino Acids by the Uryl‐Pendant Hydroxyphenyl‐Binol Ketone

The hydroxyphenyl chiral ketone, (S)‐ 3 , reacts with D ‐amino acids bearing hydrophobic side chains exclusively over the L ‐amino acids in a two‐phase liquid–liquid extraction, and thus acts as a highly stereoselective extractant. Calculations for the energy‐minimized structures for the imine diastereomers and the comparison of the selectivities with other phenyl ketones, (S)‐ 4 and (S)‐ 5 , demonstrate that the hydrogen bond between the carboxylate group and the phenolic hydroxyl group contributes to the remarkable enantioselectivities. The multiple hydrogen bonds present in the imine of (S)‐ 3 reinforce the rigidity, and results in the difference between the stabilities of the imine diastereomers. The imine could be hydrolyzed in methanolic HCl solution, and the extraction of the evaporated residues revived the organic layer of (S)‐ 3 , which could enter into a new extractive cycle and leaves the D ‐amino acid with enantiomeric excess (ee) values of over 97 % in the aqueous layer.     

Applications of Unnatural Amino Acids in Protease Probes

Since proteases are involved in a wide range of physiological and disease states, the development of novel tools for imaging proteolytic enzyme activity is attracting increasing interest from scientists. Peptide substrates containing proteinogenic amino acids are often the first line of defining enzyme specificity. This Minireview outlines examples of major recent advances in probing proteases using unnatural amino acid residues, which greatly expands the possibilities for designing substrate probes and inhibitory activity‐based probes. This approach already yielded innovative probes that selectively target only one active protease within the group of enzymes exhibiting similar specificity both in cellular assays and in bioimaging research.     

一种新型主客体荧光探针对人体氨基酸的识别研究

本文研究了环戊基全取代六元瓜环(CyP₆Q[6])与2-(β-吡啶)-1H-咪唑[4,5-f][1,10]菲咯啉盐酸盐(即Ar-IPHS)的主客体相互作用,并利用CyP₆Q[6]可使Ar-IPHS产生强烈荧光的性质制备了Ar-IPHS@CyP₆Q[6]荧光探针用以识别氨基酸。结果表明,探针可高效识别甘氨酸、赖氨酸、甲硫氨酸,检出限分别为2.27×10^-6、1.359×10^-5和1.690×10^-5mol/L。     

Biocatalysis with Unnatural Amino Acids: Enzymology Meets Xenobiology

The goal of xenobiology is to design biological systems endowed with unusual biochemical functions, whereas enzymology concerns the study of enzymes, the workhorses of biocatalysis. Biocatalysis employs enzymes and organisms to perform useful biotransformations in synthetic chemistry and biotechnology. During the past few years, the effects of incorporating noncanonical amino acids (ncAAs) into enzymes with potential applications in biocatalysis have been increasingly investigated. In this Review, we provide an overview of the effects of new chemical functionalities that have been introduced into proteins to improve various facets of enzymatic catalysis. We also discuss future research avenues that will complement unnatural mutagenesis with standard protein engineering to produce novel and versatile biocatalysts with applications in synthetic organic chemistry and biotechnology.     

A Sensitive and Selective Fluorescent Probe for Cysteine Based on a New Response‐Assisted Electrostatic Attraction Strategy: The Role of Spatial Charge Configuration

A new strategy for fast fluorescent detection of cysteine (Cys), based on a response‐assisted electrostatic attraction, is demonstrated. By utilizing this strategy, we designed and synthesized three fluorescent probes for the specific detection of Cys under actual physiological conditions. The probe m‐ CP , a coumarin fluorophore conjugated with a substituted methyl pyridinium group through an unsaturated ketone unit, showed highly selective and sensitive detection for cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH). The kinetic analysis indicated that the sensing process was highly accelerated (a response time less than 1 min) by the response‐assisted electrostatic attraction. More importantly, control experiments with isomeric probes first demonstrated that the spatial charge configuration of the probe played an important role in Cys‐preferred selectivity and kinetic rate acceleration. Furthermore, the practical utility of the probe m‐ CP in the fluorescent labeling of Cys residues within proteins was demonstrated. Finally, these probes were employed in living cell imaging with HeLa cells, in which it displayed satisfactory cell permeability and enabled us to distinguish active thiols in the cytoplasm, nucleus, and mitochondria.     

Insight into the Mechanism of Hydrogenation of Amino Acids to Amino Alcohols Catalyzed by a Heterogeneous MoOx‐Modified Rh Catalyst

Hydrogenation of amino acids to amino alcohols is a promising utilization of natural amino acids. We found that MoO_x‐modified Rh/SiO₂ (Rh–MoO_x/SiO₂) is an efficient heterogeneous catalyst for the reaction at low temperature (323 K) and the addition of a small amount of MoO_x drastically increases the activity and selectivity. Here, we report the catalytic potential of Rh–MoO_x/SiO₂ and the results of kinetic and spectroscopic studies to elucidate the reaction mechanism of Rh–MoO_x/SiO₂ catalyzed hydrogenation of amino acids to amino alcohols. Rh–MoO_x/SiO₂ is superior to previously reported catalysts in terms of activity and substrate scope. This reaction proceeds by direct formation of an aldehyde intermediate from the carboxylic acid moiety, which is different from the reported reaction mechanism. This mechanism can be attributed to the reactive hydride species and substrate adsorption caused by MoO_x modification of Rh metal, which results in high activity, selectivity, and enantioselectivity.     

Pattern Recognition of Amino Acids in Wines

New synthesized fatty acid amides (N‐(2‐(piperidin‐1‐yl)ethyl)oleamide, and N‐(2‐(pyrrolidin‐1‐yl)ethyl)oleamide) were used for the design of stochastic sensors based on nanographene paste. The stochastic sensors were used for pattern recognition of four amino acids: L‐histidine, L‐tyrosine, L‐ornithine, and L‐lysine in wines. The pattern recognition was performed based on the signatures recorded for each of the amino acids. The limits of determination allow the assay of amino acids in wine at very low concentrations faster, reliable, and more cost effective than other methods proposed to date.     

Highly Enantioselective Zinc/Amino Alcohol‐Catalyzed Alkynylation of Aldehydes

Meeting the challenge : The zinc/amino alcohol catalyzed enantioselective addition of terminal alkynes to aldehydes is effective with both phenylacetylene and methyl propiolate, leading to chiral secondary propargyl alcohols with very high enantioselectivity (see scheme).