Novel fluorescence quenching group

Abstract

The invention discloses a novel fluorescence quenching group, relates to the crossing field of biochemistry and material science, realizes ultra-efficient quenching (greater than or equal to 95%) through an intramolecular charge transfer mechanism, and has the advantages of high stability and low cost. According to the novel fluorescence quenching group, through the synergistic effect of a polycyclic aromatic hydrocarbon core structure (such as pyrene), an electron withdrawing group (such as nitryl) and an electron donating group (such as amino), the ultra-efficient fluorescence quenching effect is achieved, experimental data shows that the performance of the novel fluorescence quenching group is remarkably superior to that of DABCYL and BHQ series, and the novel fluorescence quenching group can be widely applied to the fields of molecular diagnosis, in-vivo imaging and precise medical treatment and has a good application prospect. The quenching efficiency of the compound on common fluorescent dyes (such as FAM and Cy5) reaches up to 98.2%, and the quenching efficiency is obviously superior to that of a traditional quenching group DABCYL (72.3%) and BHQ-1 (83.5%); the high-efficiency quenching performance is mainly attributed to the strong electron cloud density and intramolecular charge transfer mechanism of polycyclic aromatic hydrocarbon, and excited electrons of fluorescent molecules can be rapidly captured, so that the fluorescence intensity is obviously reduced.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biochemistry and materials science, specifically relating to a novel fluorescence quenching group based on polycyclic aromatic hydrocarbon derivatives, its preparation method, and its application in molecular beacons, fluorescence resonance energy transfer (FRET) probes, biosensors, and medical imaging. Background Technology

[0002] Fluorescence quenching groups have important applications in molecular biology, biosensing, and medical imaging.

[0003] Traditional fluorescence quenching groups (such as DABCYL and BHQ) have the following limitations:

[0004] 1. Low quenching efficiency: DABCYL has a quenching efficiency of only 60-70% for long-wavelength dyes (such as Cy5);

[0005] 2. Environmental sensitivity: BHQ is prone to aggregation and inactivation under physiological salt ion conditions;

[0006] 3. Limited functionality: Lack of modular design for specific applications.

[0007] This invention proposes a novel quenching group based on polycyclic aromatic hydrocarbon derivatives through molecular structure innovation, thereby solving the aforementioned technical bottlenecks. Summary of the Invention

[0008] This invention provides a novel fluorescence quenching group, characterized by high quenching efficiency, high selectivity, high stability, and multifunctionality. Through specific chemical structures and modifications, this quenching group can significantly improve quenching efficiency and is suitable for a variety of fluorescent molecules and experimental conditions.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] 1. Molecular design principles

[0011] Design optimization via quantum chemical calculations (DFT, B3LYP / 6-31G*):

[0012] 1) Core structure selection: The conjugated π system of pyrene can provide a strong electron cloud density. Theoretical calculations show that its LUMO energy level (-2.1eV) is significantly lower than that of DABCYL (-1.5eV), making it more likely to accept excited state electrons from fluorescent molecules;

[0013] 2) Charge transfer regulation: Nitro groups (Hammett constant σ = 1.24) are introduced at positions 2 and 11 of pyrene, and amino groups (σ = -0.66) are introduced at positions 5 and 8, forming intramolecular charge transfer channels and improving quenching efficiency by 40%.

[0014] 3) Connector arm optimization: The PEG6 chain can reduce steric hindrance, increasing the distance between the quencher group and the fluorescent molecule. ( within the radius).

[0015] 2. Innovation in Synthetic Pathways

[0016] A step-by-step region-selective modification strategy is adopted:

[0017] 1) Selective modification of the α-position (C2 / C11) of pyrene via nitration;

[0018] 2) Utilizing Pd / C catalytic hydrogenation to reduce nitro groups to amino groups avoids the side reactions of the traditional Sn / HCl reduction method;

[0019] 3) The PEG linker is coupled via EDC / NHS “click chemistry” with a reaction yield ≥90%.

[0020] 3. Performance advantages

[0021] Verification was achieved through comparative experiments (see Table 1 for details):

[0022] 1) Quenching efficiency: The quenching rate for FAM reaches 98.2% (BHQ-1 is 83.5%);

[0023] 2) Anti-interference: Stability retention rate in 10% fetal bovine serum >95% (DABCYL <70%);

[0024] 3) Multifunctionality: It can be improved by replacing the PEG chain length (PEG3-PEG) 12 It can be adapted to different application scenarios.

[0025] 4. Application Scenarios Expansion

[0026] 1) Ultrasensitive nucleic acid detection: Combined with the CRISPR-Cas12a system, the detection limit is as low as 0.01 copies / μL;

[0027] 2) Live-cell dynamic imaging: Real-time monitoring of mitochondrial pH in HeLa cells (response time < 1 second);

[0028] 3) Multimodal diagnosis and treatment integration: Integrating near-infrared II (NIR-II) fluorescence and photothermal therapy functions.

[0029] 4. Theoretical basis and innovation of molecular design

[0030] The core innovation of this invention lies in solving the problems of low efficiency and poor stability of traditional quenching groups through the synergistic effect of polycyclic aromatic hydrocarbon derivatives and charge-regulating groups. The specific design principle is as follows:

[0031] 1) Verification by quantum chemical calculations

[0032] The electronic structures of three polycyclic aromatic hydrocarbons, pyrene, perylene, and anthracene, were calculated using density functional theory (DFT, B3LYP / 6-31G*).

[0033] 2) The LUMO level of pyrene is -2.1 eV, which is significantly lower than that of DABCYL (-1.5 eV), indicating its stronger electron trapping ability;

[0034] 3) Charge transfer pathway: Introducing nitro groups at positions 2 and 11 of pyrene (σ = 1.24) and amino groups at positions 5 and 8 (σ = -0.66) forms intramolecular charge transfer channels (ICT). Theoretical calculations show that the electron transfer rate is increased to 5 × 10⁻⁶. 12 s -1 (The traditional group is 3×10) 11 s -1 ).

[0035] 2. Optimized design of the connecting arm

[0036] Verification was performed using molecular dynamics simulations (MD, AMBER force fields):

[0037] 1) The PEG6 chain can stabilize the distance between the quenching group and the fluorescent molecule at a certain level. ( Within the radius), while alkyl chains (C 12 Due to hydrophobicity, aggregation is easily caused, with distance fluctuations reaching [amount missing].

[0038] 2) Environmental responsiveness enhancement: Introducing pH-responsive groups (such as carboxyl groups) and light-responsive groups (such as azobenzene) to achieve multifunctional regulation:

[0039] 3) pH response: In an acidic environment (pH 5.0), the carboxyl group is protonated, and the quenching efficiency is increased by 10%;

[0040] 4) Photoresponse: Under ultraviolet light (365nm) irradiation, azobenzene undergoes cis-trans isomerization, and the quenching efficiency can be reversibly adjusted (ΔQ = 15%).

[0041] 5. Technological innovation in synthetic routes

[0042] The synthesis method of this invention employs a stepwise regioselective modification strategy, overcoming the bottlenecks of traditional processes:

[0043] 1) Optimization of the synthesis of pyrene with controllable core structure;

[0044] Raw materials: benzene, AlCl3, acetic anhydride; reaction conditions: 110℃, nitrogen protection, reaction time 8 hours; yield: 82% (65% in conventional process), purity ≥99% (HPLC).

[0045] 2) Restricted selective nitrification and reduction

[0046] Nitration: Fuming nitric acid (HNO3 / H2SO4 = 1:3) is used to selectively modify the α-position (C2 / C11) of pyrene, avoiding side reactions at the β-position; yield: 75%, purity: 98% (conventional method yields 60%, containing 20% ​​byproducts). Reduction: Pd / C catalytic hydrogenation is used instead of Sn / HCl reduction to avoid the destruction of the amino group by the acidic environment; yield: 88% (conventional method yields 70%).

[0047] 3) High-efficiency coupling of connecting arms

[0048] EDC / NHS coupling: Reaction conditions: room temperature, pH 7.4, DMF solvent; Yield: 92%, purity: 98.5% (compared to 80% yield by conventional esterification method).

[0049] Compared with the prior art, the beneficial effects of the present invention are:

[0050] 1. High-efficiency quenching performance

[0051] The novel fluorescence quenching group of this invention achieves ultra-high efficiency fluorescence quenching through the synergistic effect of a polycyclic aromatic hydrocarbon core structure (such as pyrene) and electron-withdrawing groups (such as nitro) and electron-donating groups (such as amino). Experimental data show that its quenching efficiency against common fluorescent dyes (such as FAM and Cy5) is as high as 98.2%, significantly better than traditional quenching groups DABCYL (72.3%) and BHQ-1 (83.5%). This high efficiency in quenching is mainly attributed to the strong electron cloud density and intramolecular charge transfer mechanism of polycyclic aromatic hydrocarbons, which can quickly capture excited-state electrons of fluorescent molecules, thereby significantly reducing fluorescence intensity. In addition, by adjusting the number and position of electron-withdrawing and electron-donating groups, the quenching efficiency can be further optimized, enabling it to exhibit excellent performance in different application scenarios.

[0052] 2. High stability and anti-interference ability

[0053] The novel fluorescence quenching group of this invention exhibits extremely high stability and anti-interference properties in complex environments. Experimental results show that after incubation in 10% fetal bovine serum for 24 hours, its quenching efficiency remains above 95%, while the stability of traditional quenching groups DABCYL and BHQ-1 decreases to 68.1% and 89.2%, respectively. This high stability is mainly attributed to the chemical inertness of the polycyclic aromatic hydrocarbon core structure and the spatial shielding effect of the PEG linker arm, which can effectively resist interference from salt ions, proteins, and other interfering substances in the environment. In addition, the novel quenching group of this invention maintains stable quenching performance within the pH range of 3-11, making it suitable for various physiological and experimental conditions and further broadening its application scope.

[0054] 3. Multifunctionality and broad application prospects

[0055] The novel fluorescence quenching group of this invention not only possesses the characteristics of high efficiency quenching and high stability, but also has multifunctionality and broad application prospects; by replacing the length of the PEG linker arm (e.g., PEG3-PEG), 12 This invention allows for flexible adjustment of the distance between the quenching group and the fluorescent molecule, thus adapting to different fluorescence resonance energy transfer (FRET) probe and molecular beacon designs. Furthermore, by introducing specific recognition groups (such as biotin and antibodies) and responsive groups (such as pH-responsive and photoresponsive groups), highly selective detection of specific biomolecules and multifunctional applications can be achieved. For example, in molecular beacons, the novel quenching group of this invention can achieve highly specific differentiation of single-base mismatches, with a detection sensitivity of 0.1 nM. In medical imaging, when combined with near-infrared fluorescent dyes (such as ICG), it can achieve high-contrast targeted imaging in vivo. These advantages make the novel fluorescence quenching group of this invention promising for broad applications in molecular diagnostics, biosensing, in vivo imaging, and precision medicine.

[0056] Summarize:

[0057] The novel fluorescence quenching group of this invention achieves a balance of high efficiency, high stability, and multifunctionality through innovative molecular design and optimized synthetic pathways. Its superior performance significantly surpasses that of traditional quenching groups, providing a powerful tool for the further development of fluorescence technology and possessing significant scientific and application value. Attached Figure Description

[0058] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0059] Figure 1 This is a comparative diagram of the molecular structure of the present invention;

[0060] Figure 2 This is a flowchart of the synthesis route of the present invention;

[0061] Figure 3 This is a schematic diagram of the molecular dynamics simulation of the present invention;

[0062] Figure 4 This is a schematic diagram of the pH response mechanism of the present invention. Detailed Implementation

[0063] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0064] Please see Figure 1 and Figure 2 ;

[0065] Example 1: Synthesis and Characterization of Novel Quenching Groups

[0066] Step 1: Pyrene Core Synthesis

[0067] Raw materials: benzene (10mL), AlCl3 (2g), acetic anhydride (1mL);

[0068] Reaction: Stirred at 110℃ for 8 hours, and monitored the reaction progress by HPLC;

[0069] Product: White crystalline pyrene (yield 82%), mp 150-152℃.

[0070] Step 2: Synthesis of Nitropyrene

[0071] Ingredients: Pyrene (1g), fuming nitric acid (5mL), concentrated sulfuric acid (15mL);

[0072] Reaction: quenched in a 50°C water bath for 3 hours, then quenched with ice water;

[0073] Product: Yellow solid pyrene-NO2 (yield 75%), ESI-MS m / z = 292.1 [M+H] + .

[0074] Step 4: Synthesis of aminopyrene

[0075] Ingredients: Pyrene-NO2 (0.5g), 10% Pd / C (50mg), hydrazine hydrate (2mL);

[0076] Reaction: Stirred at room temperature for 6 hours under H2 (1 atm);

[0077] Product: Pale yellow solid pyrene-NH2 (yield 88%) 1 H NMR(CDCl3)δ6.8-7.2(m,8H,Ar-H),4.1(s,2H,-NH2).

[0078] Step 4: Introduction of PEG Connector Arm

[0079] Raw materials: Pyrene-NH2 (0.2g), PEG6-COOH (0.5g), EDC (0.1g), NHS (0.06g);

[0080] Reaction: Stirred in DMF at room temperature for 12 hours, then purified by silica gel column chromatography.

[0081] Product: colorless oily substance pyrene-NH-PEG6-COOH (yield 92%), HPLC purity 98.5%.

[0082] Example 2: Construction and Detection of Molecular Beacons

[0083] Probe design:

[0084] Sequence: 5'-FAM-GATCGATCGATARGETTCGATCGATC-[quencher group]-3' (TARGET is a 20nt complementary region);

[0085] Annealing conditions: 95℃ for 5 minutes, then slowly cool to 25℃.

[0086] Performance testing:

[0087] Target DNA concentration gradient: 0.01-100 nM;

[0088] Detection system: Real-time fluorescence PCR instrument (excitation 485nm, emission 520nm);

[0089] Results: Detection limit 0.05 nM (S / N = 3), linear range 0.1–50 nM (R0). 2 =0.998).

[0090] Comparative Example 1: Comparison of Traditional Quenching Groups

[0091]

[0092] Conclusion: This invention is significantly superior to traditional products in terms of efficiency, stability and cost.

[0093] Example 3: Performance Comparison of Different Core Structures

[0094] Step 1: Raw materials for the synthesis of perylene core quenching groups: perylene (1g), concentrated sulfuric acid (15mL), fuming nitric acid (5mL);

[0095] Reaction: Nitrification at 60℃ for 4 hours produces dinitroperylene;

[0096] Reduction: H2 / Pd catalysis, producing diaminoperylene;

[0097] Connector arm: Introduced with a PEG6 chain;

[0098] Product: Perylene-NH-PEG6-COOH (yield 85%).

[0099] Performance testing:

[0100] Core Structure Quenching efficiency (FAM) Serum stability (24h) pyrene 98.2％ 95.4％ Peripheral 94.5％ 89.2％ Anthracene 87.3％ 78.6％

[0101] Conclusion: The pyrene core has the best overall performance and is suitable for high-sensitivity detection.

[0102] Example 4: The effect of connecting arm length on quenching efficiency

[0103] Step 1: Synthesize quenching groups with different PEG chain lengths

[0104] PEG3, PEG6, PEG 12 Each chain incorporates a pyrene core;

[0105] Products: pyrene-NH-PEG3-COOH, pyrene-NH-PEG6-COOH, pyrene-NH-PEG 12 -COOH.

[0106] Performance testing:

[0107]

[0108] Conclusion: The PEG6 chain length achieves the best balance between quenching efficiency and spatial adaptability.

[0109] Example 5: Construction of pH-responsive quenching groups

[0110] Step 1: Introduce pH-responsive groups

[0111] Raw materials: Pyrene-NH2 (0.5g), Carboxyl-PEG6 (0.8g);

[0112] Reaction: EDC / NHS coupling generates pyrene-NH-PEG6-COOH (containing a carboxyl group);

[0113] Functional verification: Quenching efficiency was tested in different pH buffer solutions.

[0114] Test results:

[0115] pH Quenching efficiency (FAM) 5.0 99.1％ 7.4 98.2％ 9.0 96.5％

[0116] Conclusion: Protonated carboxyl groups enhance charge transfer and improve quenching efficiency under acidic conditions.

[0117] Experimental conclusion:

[0118] 1. Core structure selection: Pyrene is significantly superior to perylene and anthracene in quenching efficiency (98.2%) and stability (95.4%);

[0119] 2. Linker arm optimization: The PEG6 chain length stabilizes the distance between the quencher group and the fluorescent molecule. Efficiency reached 98.2%;

[0120] 3. Functional expansion: The pH-responsive quenching group has an efficiency of up to 99.1% in acidic environments, making it suitable for tumor microenvironment detection.

[0121] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A novel fluorescence quenching group, characterized in that, include: Core structure: Polycyclic aromatic hydrocarbon derivatives, selected from pyrene, perylene, anthracene or their chemically modified derivatives; Functional modification group: includes at least one electron-withdrawing group (selected from nitro, cyano, sulfonic acid group) and at least one electron-donating group (selected from amino, hydroxy, mercapto group), wherein the electron-withdrawing group and the electron-donating group are distributed symmetrically or asymmetrically in spatial position; Connector arm: Flexible chain structure, selected from polyethylene glycol chain (PEG), alkyl chain (C3-C4) 18 ), polyamide chains or combinations thereof, are connected to the core structure through ester bonds, amide bonds or thioether bonds.

2. The novel fluorescence quenching group according to claim 1, characterized in that: The core structure of the polycyclic aromatic hydrocarbon is pyrene, and its chemical formula is C1. 16 H 10 It has the following topology: Among them, C(2), C(5), C(8), and C(11) are the connection sites of electron-withdrawing and electron-donating groups.

3. The novel fluorescence quenching group according to claim 1, characterized in that: The electron-withdrawing group is a nitro (-NO2) group, which is attached to the C(2) and C(11) sites of the pyrene core; the electron-donating group is an amino (-NH2) group, which is attached to the C(5) and C(8) sites.

4. The novel fluorescence quenching group according to claim 1, characterized in that: The connecting arm is a polyethylene glycol chain (PEG6, molecular weight 300 Da), which is connected to the C(1) site of the pyrene core via an ester bond (-O-CO-).

5. A novel fluorescence quenching group according to claim 1, characterized in that: Among them, quenching Efficiency ≥95% (based on FAM fluorescent dyes), selectivity error ≤3%, and stability retention ≥90% within the pH range of 3-11.

6. A method for preparing the novel fluorescence quenching group according to claims 1-5, characterized in that, Includes the following steps: Step 1: Polycyclic Aromatic Hydrocarbon Core Synthesis Using benzene as a raw material, naphthalene is generated through Friedel-Crafts alkylation, and then further pyrene is generated through Diels-Alder cyclization. Reaction conditions: AlCl3 catalysis, toluene solvent, nitrogen protection, temperature 110±5℃, reaction time 8 hours; Step 2: Electron-withdrawing group modification Pyrene was dissolved in concentrated sulfuric acid, and fuming nitric acid (HNO3 / H2SO4 volume ratio 1:3) was added dropwise. The reaction was carried out at 50°C for 3 hours to produce dinitropyrene (pyrene-NO2). Step 3: Modification with electron-donating groups Pyrene-NO2 was dissolved in ethanol, and hydrazine hydrate (N2H2·H2O) and Pd / C catalyst were added. The reaction was carried out under a hydrogen atmosphere for 6 hours to produce diaminopyrene (pyrene-NH2). Step 4: Connecting arm introduction Pyrene-NH2 and PEG-COOH (carboxylated polyethylene glycol, molecular weight 300 Da) were dissolved in DMF, and EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) were added. The mixture was reacted at room temperature for 12 hours to generate the target product pyrene-NH-PEG6-COOH. Step 5: Purification and Characterization The product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20:1), and then purified by... 1 Purity was verified to be ≥98% by ¹H NMR (400MHz, CDCl₃), ESI-MS, and HPLC (C18 column, acetonitrile / water = 70:30).

7. A novel fluorescence quenching group according to claim 6, characterized in that: The molecular beacon using the novel fluorescence quenching group described in claims 1-5 is characterized in that the molecular beacon comprises the following parts: Fluorescent reporter groups: selected from FAM (carboxyfluorescein), Cy5, and HEX; Stem-loop structure: a single-stranded DNA with a length of 25±2 base pairs, the sequence being 5'-GATCGATC-[target complementary region]-GATCGATC-3'; Quenching group: The novel fluorescence quenching group according to claims 1-5 is linked to the 3' end of the DNA strand via a thiophosphate bond.

8. A novel fluorescence quenching group according to claim 7, characterized in that: Its detection sensitivity reaches 0.1 nM (signal-to-noise ratio ≥3), and its specificity can distinguish single base mismatches.

9. A FRET probe using the novel fluorescence quenching group described in claims 1-5, characterized in that, The probe includes: Fluorescent donor: Green fluorescent protein (GFP) or mCherry; Fluorescent acceptor: the novel fluorescence quenching group as described in claims 1-5; Connection method: GFP is expressed by fusing it with a quenching group through genetic engineering, with a distance of 5±1nm between them.

10. The FRET probe according to claim 9, characterized in that, Its FRET efficiency is ≥85%, and its dynamic detection range covers 1pM-100nM.

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