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Fluorescent amino acids (FlAAs) have become an important chemical tool because they can be used to construct fluorescent macromolecules such as peptides and proteins without destroying their natural biomolecular properties. Specially designed fluorescent and fluorogenic amino acids, boasting distinct photophysical traits, are utilized for tracking protein-protein interactions in real-time and imaging nanoscale phenomena with exceptional spatial precision. After absorbing the radiant energy of a specific frequency, the fluorescent substance molecule transitions from the ground state to any vibration energy level of the first electronic excited state (or higher excited state). In the solution, the excited state molecules collide with the solvent molecules, lose part of the energy in the form of heat, and return to the lowest vibrational energy level of the first electronic excited state (no radiation transition). Then, in the form of radiation to deactivate any vibrational level that transitions to the electronic ground state, fluorescence will be generated.
Amino acids are a general term for a class of organic compounds containing amino and carboxyl groups. They are the basic building blocks of biologically functional macromolecular proteins and the basic substances of animal nutritional proteins. Fluorescent amino acids are modified forms of natural amino acids that contain chemical groups that emit light when exposed to certain wavelengths. These modifications can be made to the side chains or backbones of amino acids, allowing researchers to introduce fluorescence into specific regions of the protein. Tryptophan (try), tyrosine (Tyr) and phenylalanine (PHE) are the only fluorescent components among natural amino acids. They can be measured using fluorescence analysis, which has the advantages of high sensitivity and good selectivity. For example, when 2-mercaptoethanol is used as a reducing agent, o-phthalaldehyde and amino acid compounds can form strong fluorescent substances through cyclization and condensation reactions. The optimal excitation wavelength (λex) of this substance is 340 nm, and the optimal emission wavelength (λem) is 450 nm. Furthermore, the fluorescence intensity is directly proportional to the amino acid concentration.
Fig. 1. Fluorescent amino acids for chemical biology (Nat Rev Chem. 2020, 4(6): 275-290).
Unnatural fluorescent amino acids (FlAAs) have become valuable tools in the field of chemical biology, providing a less destructive method of labeling fluorescently labeled peptides and proteins. Due to their small size and similarity to natural amino acids, these FlAAs can fluorescently label macromolecules while retaining their functionality and native structure with minimal interference. The expansion of the FlAA toolbox, especially in the early 2000s, was driven by the development of new FlAAs with unique optical properties. These properties include environmental responsiveness, sensitivity to metal chelation, tunable fluorescence emission, and extended fluorescence lifetime. By incorporating these FlAAs into peptides via solid-phase peptide synthesis (SPPS) or genetically encoding them into larger proteins, researchers can create fluorescent biomolecules that closely mimic native structures. This capability opens new avenues for biological research, facilitating real-time studies of protein conformational changes, protein-protein interactions, and cellular activities.
In recent years, significant progress has been made in the development of synthetic strategies to tailor the chemical structure of FlAA. Technologies such as multicomponent reactions, metal-catalyzed reactions, photoinduced transformations, and bioorthogonal chemistry have played a crucial role in enhancing the design and synthesis of novel FlAAs. These advances have accelerated the creation of a variety of functionally improved FlAA, allowing researchers to explore new possibilities in fluorescent labeling and biomolecule research.
The use of fluorescent probe molecules at the cellular level to detect the content and changes of intracellular substances has a very important value for the development of the human level. The relevant mechanisms for detecting thiol amino acids by fluorescent probes mainly include Michael addition reaction, aldehyde cyclization reaction, sulfonamide and sulfonyl lipid cleavage reaction, etc.
On the other hand, synthesizing a fluorescent amino acid can not only be used directly in the synthesis of peptides, to prepare fluorescently labeled polypeptides, but also structurally it can induce the peptides to form a stable secondary structure, so that the obtained fluorescent peptides have certain biological activities. As a probe, it can be directly used in the study of the interaction with proteins and has great practical application prospects. For example, a synthetic alternative that minimizes the impact of fluorescent labeling is to embed fluorescent amino acid into the peptide sequence (to avoid modification of polar groups such as amines, carboxylic acids, and thiols, which may be critical for its biological activity) or optimize appropriate spacers. These methods rely on the chemical robustness and flexibility of SPPS, which enables the efficient preparation of highly diverse peptides, including those incorporating non-native fluorescent amino acids. Currently, fluorescent amino acids have many applications in the field of preparation and research of bioactive fluorescent peptides.
Fig. 2. Cell-selective labeling of proteomes with azidonorleucine (Nat Chem Biol. 2009, 5(10): 715-7).
Bacterial cells contain two main types of macromolecules assembled from amino acids, namely proteins, which are composed of L-amino acids, and peptidoglycan, which contains both L-amino acids and D-amino acids. Peptidoglycan is a complex polymer that forms the cell wall of bacteria and coordinates several important processes, including cell growth and division. The biological importance of peptidoglycan makes it a target for many antibiotics, and peptidoglycan biosynthesis has become an extensive area of research for the discovery of new antibacterial drugs. The discovery of fluorescent D-amino acids (FDAAs) provides researchers in the field with non-invasive probes to visualize key steps in the peptidoglycan biosynthesis process in bacterial cells. Exploiting the inherent promiscuity of taxonomically diverse bacteria by incorporating D-amino acids as peptidoglycan metabolites, modified D-amino acids have been used to specifically label sites of new cell wall growth in real time. Currently, fluorescent D-amino acids have three main applications in bacteria:
BOC Sciences offers a full line of fluorescent amino acids, including a variety of fluorophores, linkers and amino acid derivatives. Whether you are looking for specific fluorescent tags for protein labeling, fluorescent amino acids for peptide synthesis, or custom-designed fluorescent probes for imaging studies, we have the expertise and resources to meet your needs. All fluorescent amino acids we supply are manufactured using the highest quality materials and state-of-the-art production processes to ensure purity, consistency and performance. Each product undergoes rigorous quality control testing to ensure its effectiveness and reliability, giving customers the confidence to conduct precise and accurate research. If you are interested in our fluorescent amino acids, please contact us for more product information.
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