Luminescent oligopeptides based on natural amino acids, methods of preparation, use and virtual screening
The preparation of natural amino acid oligopeptides through quantum chemical calculations and solid-phase synthesis has solved the problems of biocompatibility and luminescence performance regulation, realized the biological application of highly efficient long-wavelength luminescent materials, and promoted the development of a new generation of bioluminescent materials.
Patent Information
- Application Number
- CN202610665323.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-14
- Publication Date
- 2026-08-25
AI Technical Summary
Existing luminescent materials suffer from poor biocompatibility, large molecular size affecting biological processes, and unclear luminescence performance regulation mechanisms in biological applications. Traditional trial-and-error material development is inefficient.
By employing a virtual screening method based on quantum chemical calculations and combined with a solid-phase synthesis strategy, oligopeptides composed of natural amino acids were prepared. Spatial conjugation was formed through amide bonds, which regulated the strength of charge transfer within and between oligopeptide chains, thereby obtaining highly efficient long-wavelength luminescence properties.
The efficient synthesis of biocompatible oligopeptide materials has been achieved, which possess long-wavelength luminescence properties in the range of 610 nm to orange light. These materials can be applied to bioimaging and life process monitoring, improving the efficiency and accuracy of material development.
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Figure CN122628136A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of weakly interacting organic light-emitting materials composed of natural amino acids, and specifically to the preparation and characterization of light-emitting oligopeptides screened based on quantitative calculations. Background Technology
[0002] Light is a core pathway for life to obtain energy and perceive the external environment, and luminescent materials have wide applications in biomedicine, optoelectronic devices, and other fields. Traditional photophysics theory holds that the luminescent performance of organic luminescent materials depends on the delocalized π-electron structure under the through-bond conjugation (TBC) mechanism. Based on this mechanism, two generations of mature fluorescent dyes have been developed: first-generation fused-ring aromatic hydrocarbon dyes and second-generation green fluorescent protein (GFP) dyes. However, the first-generation dyes suffer from biotoxicity and poor biocompatibility due to their fused-ring aromatic hydrocarbon structure, while the second-generation fluorescent dyes have key problems such as excessively large molecular size that can interfere with physiological processes in vivo and thus the monitoring process. Therefore, the application of these two generations of dyes in bioimaging and other scenarios is limited.
[0003] At the beginning of this century, anomalous luminescence phenomena in nonvalently conjugated structures were discovered, such as the blue light emission behavior in natural or synthetic polymers like starch, polyamide amines, and polyesters. This unique luminescence phenomenon is called cluster luminescence. In recent years, in-depth research on cluster luminescence has given rise to a novel through-space conjugation (TSC) luminescence mechanism, breaking the dependence of traditional luminescent materials on delocalized π-electron structures and providing a new direction for the development of novel luminescent materials. Nonvalent weak interactions such as hydrogen bonds and π-π stacking are widespread in living systems. Oligopeptides, composed of natural amino acids, possess biocompatibility, degradability, and structural controllability. The amide bonds within their molecules have the potential to form spatial conjugation interactions, making them promising carriers for next-generation bio-friendly luminescent materials. However, current research on oligopeptide luminescent materials still faces the scientific challenge of understanding the regulatory mechanism of spatial conjugation between amide bonds.
[0004] Currently, numerous teams in the field of luminescent materials have applied quantum chemical calculations to the design and screening of high-performance luminescent materials. The aforementioned team utilized quantitative calculation results to elucidate the mechanism of cluster luminescence and spatial interaction, and applied this to guide the structural design of small-molecule and polymeric luminescent materials. Compared to traditional trial-and-error material development strategies, this virtual screening method based on quantitative calculations significantly improves the efficiency and accuracy of material development and design. Therefore, combining quantitative calculations with virtual screening to obtain oligopeptides with excellent luminescent properties can provide an efficient and universal research strategy for the rational design of bioluminescent materials.
[0005] Furthermore, although oligopeptides have been widely used as biomaterials in many fields of life sciences, such as antibacterial and drug delivery, their application as optical functional materials has not yet been reported. By regulating the structure of oligopeptides through different structural modification strategies, combined with photophysical characterization and theoretical calculations, the structure-activity relationship between the structure and spatial conjugation intensity of oligopeptides can be established, enabling long-wavelength luminescence of oligopeptides. This holds promise for promoting the development of next-generation bioluminescent materials.
[0006] Solid-phase synthesis is an effective method for achieving controllable oligopeptide sequences. Based on the spatial conjugation effect and electron delocalization theory, introducing heteroatom groups, heavy atom groups, electron-donating and electron-withdrawing groups into the side groups of oligopeptides can regulate the strength of intra- and inter-chain space charge transfer, spatial conjugation, and intersystem crossing efficiency, which can significantly alter the electronic structure. This is of great significance for preparing high-efficiency and long-wavelength weakly interacting luminescent oligopeptides. Summary of the Invention
[0007] To address the aforementioned technical problems and shortcomings in this field, this invention provides a class of luminescent oligopeptides based on natural amino acids, their preparation methods, applications, and virtual screening methods. This invention combines virtual screening with solid-phase synthesis strategies to obtain oligopeptides with highly efficient photofluorescence properties, wherein all amino acids used are derived from natural amino acids. This invention achieves precise and efficient synthesis of oligopeptides, overcoming the technical defects of poor biocompatibility and low preparation efficiency of traditional luminescent materials.
[0008] The specific technical solution of this invention is as follows: In a first aspect, the present invention provides luminescent oligopeptides based on natural amino acids, wherein the amino acid sequence is proline-tyrosine-tryptophan or proline-tryptophan-asparagine.
[0009] The above two luminescent tripeptides can be obtained by combining virtual screening with experimental synthesis and characterization.
[0010] The luminescent oligopeptides of this invention are linked by amide bonds, and there is spatial conjugation between amide motifs, between amide motifs and amino acid side groups, or between side groups within the molecule.
[0011] This invention yields two tripeptides with long-wavelength emission characteristics (emission wavelengths covering the range from yellow to orange light), whose amino acid sequences are proline-tyrosine-tryptophan and proline-tryptophan-asparagine, respectively, and whose optimal emission wavelengths are 610 nm (orange light) and 600 nm (orange light), respectively.
[0012] In a second aspect, the present invention provides a method for preparing luminescent oligopeptides based on natural amino acids as described in the first aspect. The method employs a solid-phase synthesis method, using 2-chlorotriphenylmethyl chloride (CTC) resin as a solid-phase support and 9-fluorenylmethoxycarbonyl (Fmoc) as an amino acid protecting group, and gradually linking amino acids.
[0013] In some preferred embodiments, the preparation method includes: Resin activation and connection to the first amino acid: Weigh 2-chlorotriphenylmethyl chloride resin and the first amino acid protected by 9-fluorenylmethoxycarbonyl in a reactor, add a good solvent and N,N-diisopropylethylamine (DIPEA), react for a period of time (e.g., 2-3 hours), then add methanol (HPLC grade) for end-capping (end-capping time can be half an hour, etc.), and wash (specifically, wash twice with N,N-dimethylformamide (DMF), once with methanol, once with dichloromethane (DCM), once with methanol, once with DCM, and twice with DMF). One-step deprotection: Add 20% piperidine / N,N-dimethylformamide solution (the amount can be 3 times the resin volume, etc.), purge with nitrogen (e.g., for 30 minutes, etc.) to remove the 9-fluorenylmethoxycarbonyl protecting group, and wash (specifically, wash 5 times with 2 times the resin volume of N,N-dimethylformamide). The second amino acid condensation: The second amino acid protected by 9-fluorenylmethoxycarbonyl is added to the resin, along with N,N-dimethylformamide, N,N-diisopropylethylamine and benzotriazole tetramethylurea hexafluorophosphate (HBTU). After reacting for a period of time (e.g., 30 minutes), the resin is washed (specifically, the resin can be washed 3 times with 2 times the volume of DMF). Secondary deprotection: Add 20% piperidine / N,N-dimethylformamide solution and purge with nitrogen (e.g., for 30 minutes) to remove the 9-fluorenylmethoxycarbonyl protecting group, followed by washing (specifically, washing 5 times with 2 times the resin volume of DMF). The third amino acid condensation: The third amino acid protected by 9-fluorenylmethoxycarbonyl is added to the resin, along with N,N-dimethylformamide, N,N-diisopropylethylamine and benzotriazole tetramethylurea hexafluorophosphate. After reacting for a period of time (e.g., 30 minutes), the resin is washed (specifically, the resin can be washed 3 times with 2 times the volume of DMF). Three-stage deprotection: Add 20% piperidine / N,N-dimethylformamide solution and purge with nitrogen (e.g., for 30 minutes) to remove the 9-fluorenylmethoxycarbonyl protecting group, followed by washing (specifically, washing 5 times with 2 times the resin volume of DMF). Cutting: Add cutting fluid (the amount can be 6 times the resin volume, etc.), disperse the resin with (short-time) ultrasonic assistance, and stir (at low speed) (e.g., for 2 hours, etc.), filter to remove resin, precipitate the filtrate with ice-cold anhydrous diethyl ether, and wash the precipitate with ice-cold anhydrous diethyl ether (e.g., 3 times, etc.), vacuum dry (specifically, the precipitate can be placed in a vacuum drying oven, etc.), and separate and purify.
[0014] In some preferred embodiments, the initial degree of substitution of the 2-chlorotriphenylmethyl chloro resin is 1.0 mmol / g.
[0015] In some preferred embodiments, good solvents include, but are not limited to, dichloromethane (DCM).
[0016] In some preferred embodiments, during the amino acid condensation process, the molar ratio of the added amino acid, N,N-diisopropylethylamine, benzotriazole tetramethylurea hexafluorophosphate, and resin is 3:6:3:1.
[0017] Thirdly, this invention provides the application of the luminescent oligopeptides based on natural amino acids described in the first aspect in fluorescent probes of biological systems. Furthermore, the luminescent oligopeptides of this invention can be used for bioimaging and process tracing.
[0018] The luminescent oligopeptides of this invention have broad application prospects in the field of biology. Combined with intracellular enzyme catalysis technology, they are expected to develop into the third type of organic fluorescent probe after traditional conjugated organic dyes and fluorescent proteins, with great application prospects in fields such as bioimaging and life process monitoring.
[0019] As a general inventive concept, in a fourth aspect, the present invention provides a virtual screening method for luminescent oligopeptides based on natural amino acids, comprising: A dipeptide structure library was constructed using 22 natural amino acids. Using time-dependent density functional theory as the calculation method, two functionals, ωB97XD and B3LYP-D3, were compared and calculated to screen for dipeptides with excellent optical properties (small band gap between the highest occupied orbital (HOMO) and lowest vacant orbital (LUMO), and high first excited state (S1) energy level and its corresponding oscillator strength). An amino acid motif was inserted at any position to construct a tripeptide structure library. Quantitative calculations were performed to screen for target tripeptide structures with narrow band gaps and strong oscillator strength, which were then used as candidates for luminescent oligopeptides based on natural amino acids.
[0020] The 22 natural amino acids mentioned are alanine, valine, leucine, isoleucine, meleucine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, aspartic acid, glutamine, glycine, histidine, lysine, arginine, aspartic acid, glutamic acid, pyrrolidone, and selenocysteine.
[0021] In some preferred embodiments, the virtual screening method constructs dipeptides with tryptophan as the core structural unit, whose indole group provides the basis for the formation of spatial conjugation. Tryptophan is combined with 22 natural amino acids to construct a dipeptide structure library.
[0022] Furthermore, the virtual screening method uses the band gap between the highest occupied orbit and the lowest empty orbit, as well as the first excited state energy level and its corresponding oscillator strength, as the core screening indicators.
[0023] The screening criteria for dipeptides with excellent optical properties are mainly based on band gap difference. In some preferred examples, dipeptides with excellent optical properties have a band gap of < 2 eV.
[0024] In some preferred embodiments, the target tripeptide structure with a narrow bandgap and strong oscillator strength corresponds to a bandgap ≤ 0.7 eV, and the emission wavelength of the first excited state returning to the ground state. λ S1 > 650 nm (reflecting a large first excited state energy level), oscillator strength > 2×10 -3 .
[0025] Compared to tripeptides, dipeptides have simpler structures and more uniform conformations, resulting in generally lower and less varied oscillator strengths. The oscillator strength, which truly determines luminescence intensity, only becomes significant after tripeptides form longer, more conjugated, and more stable conformations. Tripeptides have longer backbones, larger π-electron delocalization ranges, and smaller HOMO-LUMO band gaps. They also have more heteroatom interaction sites and conformational regulation space, which is beneficial for enhancing the spatial conjugation effect between side groups and amide bonds, as well as intramolecular complexation, and reducing nonradiative transition losses.
[0026] In some preferred embodiments, the virtual screening method uses a B3LYP-D3 functional for quantization calculations.
[0027] In the luminescent oligopeptides of this invention, the energy difference (i.e., band gap, Δ) between the LUMO and HOMO of the proline-tyrosine-tryptophan excited states is... E The ΔV of the proline-tryptophan-asparagine excited state is 0.2212 eV. E It is 0.5203 eV.
[0028] After virtual screening, tripeptides can be prepared according to the preparation method described in the second aspect. The correctness of the oligopeptide structure and its luminescent properties are verified through structural characterization and photophysical performance characterization to obtain luminescent oligopeptides that meet the requirements. The structural characterization can employ one or more of the following: proton / carbon nuclear magnetic resonance spectroscopy, high-resolution mass spectrometry, and elemental analysis. The photophysical performance characterization can employ one or more of the following: steady-state absorption spectroscopy, fluorescence spectroscopy, and fluorescence quantum efficiency.
[0029] This invention employs a solid-phase synthesis method to achieve controllable synthesis of oligopeptide sequences. By combining theoretical calculations and virtual screening, a closed-loop model of theoretical prediction, experimental verification, and optimization re-prediction is constructed, which significantly improves the efficiency and accuracy of material development.
[0030] Compared with the prior art, the beneficial effects of this invention are as follows: 1) In terms of materials system: For the first time, oligopeptides composed of natural amino acids are systematically studied and developed as an intrinsically luminescent functional material.
[0031] 2) In terms of development methods: a precise design path based on the combination of quantum chemical calculation and virtual screening is proposed, and a closed-loop mode of theoretical prediction-synthesis verification-result feedback-optimization and re-prediction is adopted.
[0032] 3) Material properties: Based on the spatial conjugation between amino acid residues within and between oligopeptide chains, tripeptide materials with emission wavelengths of 610 nm, 600 nm, and 574 nm were constructed using natural amino acids, realizing long-wavelength emission of oligopeptides, promoting the development of next-generation bioluminescent materials, and the synthesized materials have great potential for application in fields such as bioimaging and life process monitoring. Attached Figure Description
[0033] Figure 1 The image shows the 1H NMR spectrum of the proline-tyrosine-tryptophan prepared in Example 2.
[0034] Figure 2 The mass spectrum of proline-tyrosine-tryptophan prepared in Example 2 is shown.
[0035] Figure 3 The image shows the 1H NMR spectrum of the proline-tryptophan-asparagine prepared in Example 3.
[0036] Figure 4 The mass spectrum of proline-tryptophan-asparagine prepared in Example 3 is shown.
[0037] Figure 5 The solid-state fluorescence spectrum of the proline-tyrosine-tryptophan prepared in Example 2 is shown below. λ ex Indicates the excitation wavelength.
[0038] Figure 6 The solid-state fluorescence spectrum of proline-tryptophan-asparagine prepared in Example 3 is shown. λ ex Indicates the excitation wavelength.
[0039] Figure 7 The fluorescence excitation spectrum of proline-tyrosine-tryptophan prepared in Example 2 with varying solution concentrations, and the emission wavelength was fixed. λ em The value is 535 nm.
[0040] Figure 8 The fluorescence excitation spectrum of proline-tryptophan-asparagine prepared in Example 3 with varying solution concentrations is shown, with the emission wavelength fixed at 530 nm. Detailed Implementation
[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically specified in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer.
[0042] Example 1: Virtual screening of target tripeptides: Construction of dipeptide structure library: Using tryptophan as the core, it was combined with 22 kinds of natural amino acids to construct a dipeptide structure library.
[0043] Quantitative calculation: Time-dependent density functional theory was used as the calculation method, and two functionals, ωB97XD and B3LYP-D3, were used for comparison. The calculation results are expressed in terms of Δ E (LUMO-HOMO) The core screening criteria are the energy range between LUMO and HOMO, the S1 energy level and its oscillator strength.
[0044] Dipeptide screening: Based on the calculation results, two narrow bandgap emission dipeptides, proline-tryptophan and aspartic acid-tryptophan, were screened out. Their excited-state bandgap is narrow, and they have the potential to form long-wavelength emission.
[0045] Construction and screening of tripeptide structure library: An amino acid was inserted at any position of the two dipeptides screened above to construct a tripeptide structure library. B3LYP-D3 functional analysis was used for quantitative calculations to screen out five target tripeptides: proline-tyrosine-tryptophan, proline-tryptophan-asparagine, aspartic acid-proline-tryptophan, cysteine-proline-tryptophan, and proline-arginine-tryptophan. These five tripeptides have both a small S1 energy level and a large oscillator strength, and are predicted to have excellent long-wavelength luminescence properties.
[0046] Example 2: Synthesis of proline-tyrosine-tryptophan: Using proline, tyrosine, and tryptophan as raw materials, CTC resin was selected as the solid-phase support, and Fmoc was used as the amino acid protecting group. The amino acids were sequentially linked according to the proline-tyrosine-tryptophan amino acid sequence. The specific steps are as follows: Weigh CTC resin (CTC Resin, initial substitution degree approximately 1.0 mmol / g) and Fmoc-tryptophan-OH into a reactor. Add an appropriate amount of DCM to dissolve the amino acid, then add DIPEA. After reacting for 2-3 hours, add HPLC-grade methanol for end-capping for half an hour. Wash twice with DMF, once with methanol, once with DCM, once with methanol, once with DCM, and twice with DMF to obtain Fmoc-tryptophan-CTC Resin. Add 3 times the resin volume of 20% piperidine / DMF solution to the above resin, purge with nitrogen for 30 minutes to remove the Fmoc protecting group (ninhydrin detection is blue). Wash 5 times with 2 times the resin volume of DMF to obtain H2N-tryptophan-CTC. Resin; Add Fmoc-tyrosine-OH to the above resin, dissolve in an appropriate amount of DMF, then add DIPEA and HBTU (amino acid: DIPEA: HBTU: resin = 3:6:3:1, molar ratio). After reacting for 30 minutes, wash three times with twice the resin volume of DMF to obtain Fmoc-tyrosine-tryptophan-CTC Resin; Remove the Fmoc protecting group (ninhydrin detection is blue), wash five times with DMF to obtain H2N-tyrosine-tryptophan-CTC Resin; Add Fmoc-proline-OH to the above resin, dissolve in an appropriate amount of DMF, then add DIPEA and HBTU (amino acid: DIPEA: HBTU: resin = 3:6:3:1, molar ratio). After reacting for 30 minutes, wash three times with DMF to obtain Fmoc-proline-tyrosine-tryptophan-CTC. Resin; remove the Fmoc protecting group (indicated by ninhydrin detection in blue), wash 5 times with DMF to obtain H2N-proline-tyrosine-tryptophan-CTC Resin; add 6 times the resin volume of cutting fluid, briefly sonicate to disperse the resin, and stir at low speed for 2 hours. Filter to remove the resin, precipitate the filtrate with ice-cold anhydrous diethyl ether, wash 3 times, and vacuum dry to obtain the crude product; separate and purify the synthesized product to remove impurities and unreacted raw materials to obtain a high-purity tripeptide; verify the structure of proline-tyrosine-tryptophan using 1H NMR spectroscopy and high-resolution mass spectrometry, such as... Figure 1 , Figure 2 As shown, the structure was confirmed to be correct; its photophysical properties were characterized using fluorescence spectroscopy, such as... Figure 5 As shown, the results indicate that the optimal emission wavelength for proline-tyrosine-tryptophan in the solid state is 610 nm, successfully achieving long-wavelength luminescence of oligopeptides based on natural amino acids. Figure 7 As shown, it was observed that the emission peak exhibited a redshift and the waveform changed with increasing concentration, reaching a certain concentration (1×10⁻⁶). -3 After reaching a concentration of mol / L, the emission intensity increased sharply, exhibiting typical cluster emission characteristics, rather than a simple concentration-enhanced fluorescence effect.
[0047] Example 3: Synthesis of proline-tryptophan-asparagine: Using proline, tryptophan, and asparagine as raw materials, CTC resin was selected as the solid-phase support, and Fmoc was used as the amino acid protecting group. The amino acids were sequentially linked according to the proline-tryptophan-asparagine amino acid sequence. The specific steps are as follows: Weigh CTC resin (initial degree of substitution approximately 1.0 mmol / g) and Fmoc-asparagine-OH into a reactor. Add an appropriate amount of DCM to dissolve the amino acids, then add DIPEA. After reacting for 2-3 hours, add HPLC-grade methanol for end-capping for half an hour. Wash twice with DMF, once with methanol, once with DCM, once with methanol, once with DCM, and twice with DMF to obtain Fmoc-asparagine-CTC Resin. Add 3 times the resin volume of 20% piperidine / DMF solution to the above resin, purge with nitrogen for 30 minutes to remove the Fmoc protecting group (ninhydrin detection is blue). Wash 5 times with 2 times the resin volume of DMF to obtain H2N-asparagine-CTC. Resin; Add Fmoc-tryptophan-OH to the above resin, dissolve in an appropriate amount of DMF, then add DIPEA and HBTU (amino acid: DIPEA: HBTU: resin = 3:6:3:1, molar ratio). After reacting for 30 minutes, wash three times with twice the resin volume of DMF to obtain Fmoc-tryptophan-asparagine-CTC Resin; Remove the Fmoc protecting group (ninhydrin detection is blue), wash five times with DMF to obtain H2N-tryptophan-asparagine-CTC Resin; Add Fmoc-proline-OH to the above resin, dissolve in an appropriate amount of DMF, then add DIPEA and HBTU (amino acid: DIPEA: HBTU: resin = 3:6:3:1, molar ratio). After reacting for 30 minutes, wash three times with DMF to obtain Fmoc-proline-tryptophan-asparagine-CTC. Resin was used to remove the Fmoc protecting group (indicated by a blue ninhydrin test), and the sample was washed five times with DMF to obtain H2N-proline-tryptophan-asparagine-CTCResin. Six times the resin volume of cutting fluid was added, and the resin was dispersed using short-term ultrasonic-assisted dispersion and low-speed stirring for 2 hours. The resin was removed by filtration, and the filtrate was precipitated with ice-cold anhydrous diethyl ether. After washing three times, the crude product was vacuum dried to obtain the crude product. The synthesized product was further purified to remove impurities and unreacted raw materials, yielding a high-purity tripeptide. The structure of asparagine-tryptophan-proline was verified using 1H NMR spectroscopy and high-resolution mass spectrometry. Figure 3 , Figure 4 As shown, the structure was confirmed to be correct; its photophysical properties were characterized using fluorescence spectroscopy, such as... Figure 6As shown, the results indicate that the optimal emission wavelength for asparagine-tryptophan-proline in the solid state is 600 nm, successfully achieving long-wavelength luminescence of oligopeptides based on natural amino acids. Figure 8 As shown, with increasing concentration, its emission peak shape undergoes asymmetrical changes and a certain tailing phenomenon exists at the long-wavelength end, proving that its luminescence mechanism is cluster luminescence, which is fundamentally different from the simple concentration effect.
[0048] Furthermore, it should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.
Claims
1. A luminescent oligopeptide based on natural amino acids, characterized in that, The amino acid sequence is proline-tyrosine-tryptophan or proline-tryptophan-asparagine.
2. The method for preparing luminescent oligopeptides based on natural amino acids according to claim 1, characterized in that, A solid-phase synthesis method was adopted, using 2-chlorotriphenylmethyl chloride resin as the solid support and 9-fluorenylmethoxycarbonyl as the amino acid protecting group, and amino acids were gradually linked together.
3. The preparation method according to claim 2, characterized in that, The preparation method includes: Resin activation and connection to the first amino acid: Weigh 2-chlorotriphenylmethyl chloride resin and the first amino acid protected by 9-fluorenylmethoxycarbonyl in a reactor, add a good solvent and N,N-diisopropylethylamine, react for a period of time, add methanol to seal the end, and wash. First-stage deprotection: Add 20% piperidine / N,N-dimethylformamide solution, purge with nitrogen to remove the 9-fluorenylmethoxycarbonyl protecting group, and wash; The second amino acid condensation: The second amino acid protected by 9-fluorenylmethoxycarbonyl was added to the resin, along with N,N-dimethylformamide, N,N-diisopropylethylamine and benzotriazole tetramethylurea hexafluorophosphate. After reacting for a period of time, the mixture was washed. Secondary deprotection: Add 20% piperidine / N,N-dimethylformamide solution and purge with nitrogen to remove the 9-fluorenylmethoxycarbonyl protecting group, then wash; The third amino acid condensation: The third amino acid protected by 9-fluorenylmethoxycarbonyl was added to the resin, along with N,N-dimethylformamide, N,N-diisopropylethylamine and benzotriazole tetramethylurea hexafluorophosphate. After reacting for a period of time, the mixture was washed. Three-stage deprotection: Add 20% piperidine / N,N-dimethylformamide solution and purge with nitrogen to remove the 9-fluorenylmethoxycarbonyl protecting group, then wash; Cutting: Add cutting fluid, disperse the resin with ultrasonic assistance while stirring, filter to remove resin, precipitate the filtrate with ice-cold anhydrous diethyl ether, wash the precipitate with ice-cold anhydrous diethyl ether, vacuum dry, and separate and purify.
4. The preparation method according to claim 2 or 3, characterized in that, The initial degree of substitution of 2-chlorotriphenylmethyl chloride resin is 1.0 mmol / g.
5. The preparation method according to claim 3, characterized in that, Good solvents include dichloromethane.
6. The preparation method according to claim 3, characterized in that, During the amino acid condensation process, the molar ratio of the added amino acid, N,N-diisopropylethylamine, benzotriazole tetramethylurea hexafluorophosphate, and resin is 3:6:3:
1.
7. The application of the luminescent oligopeptide based on natural amino acids as described in claim 1 in fluorescent probes of biological systems.
8. A virtual screening method for luminescent oligopeptides based on natural amino acids, characterized in that, include: A dipeptide structure library was constructed using 22 natural amino acids. Using time-dependent density functional theory as the calculation method, two functionals, ωB97XD and B3LYP-D3, were compared and calculated to screen dipeptides with excellent optical properties. An amino acid motif was inserted at any position to construct a tripeptide structure library. Quantitative calculations were performed to screen target tripeptide structures with narrow band gaps and strong oscillator strength as candidates for luminescent oligopeptides based on natural amino acids.
9. The virtual screening method according to claim 8, characterized in that, The energy gap between the highest occupied orbit and the lowest empty orbit, as well as the energy level of the first excited state and its corresponding oscillator strength, are used as the core screening indicators.
10. The virtual screening method according to claim 9, characterized in that, Excellent optical performance with a dipeptide bandgap of < 2 eV; The target tripeptide structure with a narrow bandgap and strong oscillator strength corresponds to a bandgap ≤ 0.7 eV, and the emission wavelength of the first excited state returning to the ground state. λ S1 > 650 nm, oscillator strength > 2×10 -3 .