A rhodamine b modified lysine derivative, and a synthesis method and application thereof

Abstract

This invention discloses a rhodamine B-modified lysine derivative, its synthesis method, and its applications. The structural formula of the rhodamine B-modified lysine derivative is shown below. The invention first synthesizes Fmoc-Lys(Boc)-OtBu using commercially available Fmoc-Lys(Boc)-OH as a starting material. Then, after selectively removing the Boc protecting group from the side chain amino group, it condenses with rhodamine B to generate Fmoc-Lys(Rho)-OtBu. Finally, the tBu protecting group is removed to obtain Fmoc-Lys(Rho)-OH. The Fmoc-Lys(Rho)-OH synthesized by this invention can be loaded onto peptides using Fmoc solid-phase peptide synthesis technology for rapid and automated synthesis of rhodamine B-modified peptides.

Description

Technical Field

[0001] This invention relates to a rhodamine B-modified lysine derivative, its synthesis method, and its application, belonging to the field of polypeptide synthesis. Background Technology

[0002] Peptide fluorescent probes possess high selectivity and biocompatibility, and are easy to prepare and modify, playing a prominent role in disease detection, medical diagnosis, drug evaluation, and basic scientific research. Rhodamine B, a fluorescent group, is widely used due to its low cost, structural stability, and high fluorescence quantum yield.

[0003] Currently, Rhodamine B-modified peptides are generally obtained through side-chain condensation strategies. For example, in the article (Angew. Chem. Int. Ed. 2022, 61, e20220379), a lysine group (Fmoc-Lys(Mtt)-OH) protecting the side-chain amino group of 4-methyltriphenylmethyl (Mtt) was first introduced into the peptide sequence using a 9-fluorenemethoxycarbonyl-based solid-phase peptide synthesis technique (Fmoc-SPPS). Then, the Mtt group on the lysine side-chain amino group was removed, and Rhodamine B was condensed and coupled onto the side-chain amino group, ultimately synthesizing a Rhodamine B-modified ubiquitin protein. The above synthetic strategies require the use of orthogonally protected lysines with side-chain amino groups, such as commercially available Fmoc-Lys(Mtt)-OH and Fmoc-Lys(ivDde)-OH. The removal conditions for these orthogonally protecting groups are often incompatible with automated solid-phase peptide synthesis, making the acquisition of rhodamine-modified peptides via this method a cumbersome manual process. More importantly, in some cases, the efficiency of obtaining fluorescently labeled peptides by condensing rhodamine B with lysine side-chain amino groups is extremely low (e.g., the Lys side-chain amino group in the peptide sequence: Cys-AEEA2-Lys-AEEA2-SUMO (1-47)).

[0004] Here, we propose a rhodamine B-modified lysine derivative and its synthetic method. The lysine derivative can be directly introduced into the peptide sequence using an automated Fmoc-SPPS synthesizer. This molecule can efficiently obtain rhodamine B-modified peptides that are difficult to synthesize using side-chain condensation strategies. Summary of the Invention

[0005] This invention addresses the shortcomings of the existing technology by providing a rhodamine B-modified lysine derivative, its synthesis method, and its applications. The Fmoc-Lys(Rho)-OH synthesized in this invention can be loaded onto peptides using Fmoc solid-phase peptide synthesis technology, enabling efficient, rapid, and automated acquisition of rhodamine B-modified peptides.

[0006] The rhodamine B-modified lysine derivative of this invention, abbreviated as Fmoc-Lys(Rho)-OH, has the following structural formula:

[0007]

[0008] The present invention discloses a method for synthesizing rhodamine B-modified lysine derivatives. First, Fmoc-Lys(Boc)-OH is synthesized from commercially available Fmoc-Lys(Boc)-OH. Then, the Boc protecting group is selectively removed, and the product reacts with rhodamine B to generate Fmoc-Lys(Rho)-OtBu. Finally, the tBu protecting group is removed to obtain Fmoc-Lys(Rho)-OH. The synthetic route is shown below:

[0009]

[0010] The method for synthesizing the rhodamine B-modified lysine derivative of the present invention includes the following steps:

[0011] Step 1: Synthesis of Fmoc-Lys(Boc)-OtBu

[0012] Fmoc-Lys(Boc)-OH was dissolved in dichloromethane (DCM), and then dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) were added to the reaction system and stirred until dissolved. Finally, tert-butanol (tBu-OH) was added and the reaction was carried out overnight. After the reaction was completed, the precipitate was removed by filtration, and the resulting organic phase was concentrated under reduced pressure and purified by column chromatography to obtain Fmoc-Lys(Boc)-OtBu.

[0013] In step 1, the molar ratio of Fmoc-Lys(Boc)-OH, tBu-OH, DCC, and DMAP is 1:2:2:0.125.

[0014] Step 2: Synthesis of Fmoc-Lys(Rho)-OtBu

[0015] 2a. Dissolve Fmoc-Lys(Boc)-OtBu in a 1,4-dioxane solution of hydrogen chloride and stir for 20 minutes. After the reaction is complete, concentrate the reaction solution to obtain the crude product Fmoc-Lys-OtBu.

[0016] 2b. Rhodamine B and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) were dissolved in N,N-dimethylformamide (DMF), and N,N-diisopropylethylamine (DIEA) was added and the mixture was shaken and activated for 1 minute. The mixture was then added to a DMF solution containing the crude product Fmoc-Lys-OtBu and stirred for 2 hours. After the reaction was completed, the reaction system was diluted with water and extracted multiple times with ethyl acetate. The organic phase was collected, washed with saturated NaCl solution, dried, concentrated, and finally purified by column chromatography to obtain Fmoc-Lys(Rho)-OtBu.

[0017] In step 2a, the concentration of hydrogen chloride in the 1,4-dioxane solution of hydrogen chloride is 4M.

[0018] In step 2b, the molar ratio of crude products Fmoc-Lys-OtBu, Rhodamine B, HATU, and DIEA is 1:1.1:1.1:2.

[0019] Step 3: Synthesis of Fmoc-Lys(Rho)-OH

[0020] Fmoc-Lys(Rho)-OtBu was dissolved in DCM, and then trifluoroacetic acid (TFA) was added and stirred overnight. After the reaction was completed, the remaining TFA in the reaction system was removed by vacuum distillation to finally obtain Fmoc-Lys(Rho)-OH.

[0021] In step 3, the molar ratio of Fmoc-Lys(Rho)-OtBu to TFA is 1:60.

[0022] In step 3, the volume ratio of DCM to TFA is 1:1.

[0023] The application of the rhodamine B-modified lysine derivative of this invention is achieved by directly introducing Fmoc-Lys(Rho)-OH into the peptide sequence through an automated peptide solid-phase synthesizer, thus realizing the rapid and automated acquisition of rhodamine B-modified peptides.

[0024] Specifically, the present invention uses Fmoc-Lys(Rho)-OH and an automated peptide solid-phase synthesizer (CSBioCS136) to automatically synthesize Rhodamine B-modified SUMO(1-47), and obtains Rhodamine B-modified cR10.

[0025] The beneficial effects of this invention are reflected in:

[0026] This invention designs a lysine derivative, Fmoc-Lys(Rho)-OH, which can be used for the efficient, rapid, and automated acquisition of rhodamine B-modified peptides. Using the Fmoc-Lys(Rho)-OH provided by this invention, rhodamine B-modified SUMO(1-47) (peptide sequence: H2N-Cys-AEEA2-K(Rho)-AEEA2-Nle-ADEKPKEGVKTENNDHINLKVAGQDGSVVQFKIKRHTPLSKLMKAY-CONHNH2) can be synthesized automatically. Furthermore, the rhodamine B-modified cR10 can also be used for live-cell fluorescence imaging to detect the membrane-penetrating effect of cR10. Attached Figure Description

[0027] Figure 1 This is the proton NMR spectrum of the compound Fmoc-Lys(Boc)-OtBu.

[0028] Figure 2 This is the proton NMR spectrum of the compound Fmoc-Lys(Rho)-OtBu.

[0029] Figure 3 This is the carbon spectrum of the compound Fmoc-Lys(Rho)-OtBu.

[0030] Figure 4 This is the hydrogen spectrum of the compound Fmoc-Lys(Rho)-OH.

[0031] Figure 5 This is the carbon spectrum of the compound Fmoc-Lys(Rho)-OH.

[0032] Figure 6 This is a high-phase liquid chromatogram of the polypeptide Cys-AEEA2-Lys-AEEA2-SUMO (1-47)-CONHNH2.

[0033] Figure 7 This is the mass spectrum of the polypeptide Cys-AEEA2-Lys-AEEA2-SUMO (1-47)-CONHNH2.

[0034] Figure 8 This is a high-performance liquid chromatography (HPLC) chromatogram of the crude peptide after the condensation of Rho B with the lysine side chain of Cys-AEEA2-Lys-AEEA2-SUMO (1-47)-CONHNH2.

[0035] Figure 9 This is the mass spectrum of the polypeptide Cys-AEEA2-K(Rho)-AEEA2-SUMO (1-47)-CONHNH2.

[0036] Figure 10This is a high-performance liquid chromatography (HPLC) chromatogram of the crude peptide Cys-AEEA2-K(Rho)-AEEA2-SUMO (1-47)-CONHNH2 synthesized using Fmoc-Lys(Rho)-OH.

[0037] Figure 11 This is a high-performance liquid chromatogram of the crude peptide K(Rho)-AEEA2-cR10-G-CONH2.

[0038] Figure 12 This is the mass spectrum of the polypeptide K(Rho)-AEEA2-cR10-G-CONH2.

[0039] Figure 13 This is a laser confocal imaging image of HeLa cells after incubation with peptide K(Rho)-AEEA2-cR10-G-CONH2. Detailed Implementation

[0040] To facilitate understanding of the present invention, the implementation process of the present invention will be further described below with reference to specific embodiments. These descriptions are only for further illustrating the features and advantages of the present invention, and are not intended to limit the scope of the claims.

[0041] Example 1:

[0042] 1. Fmoc-Lys(Boc)-OH (3 g, 6.4 mmol) was added to a round-bottom flask and dissolved in 20 mL of dichloromethane (DCM). Then, dicyclohexylcarbodiimide (DCC, 2.6 g, 12.6 mmol) and 4-dimethylaminopyridine (DMAP, 97.5 mg, 0.8 mmol) were added sequentially to the round-bottom flask. Finally, tert-butanol (tBu-OH, 1.2 mL, 12.5 mmol) was added, and the reaction was carried out overnight at room temperature. After the reaction was completed, the precipitate was removed by filtration. The collected filtrate was concentrated under reduced pressure using a rotary evaporator and then purified by column chromatography to obtain compound Fmoc-Lys(Boc)-OtBu (1.8 g, yield 53%).

[0043] 2. The compound Fmoc-Lys(Boc)-OtBu (1.8 g, 3.4 mmol) was dissolved in 10 mL of 4M hydrogen chloride solution of 1,4-dioxane. After stirring for 20 minutes, the solvent was removed by rotary evaporation to obtain the crude product Fmoc-Lys-OtBu.

[0044] 3. The crude product from the previous step was dissolved in 10 mL of N,N-dimethylformamide (DMF). Rhodamine B (1.5 g, 3.1 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 1.2 g, 3.1 mmol) were then dissolved in 10 mL of DMF, and N,N-diisopropylethylamine (DIEA, 1 mL, 5.7 mmol) was added and the mixture was shaken and activated for 1 minute. The mixed solution was then added to the DMF solution containing the crude product, and the reaction was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was diluted with 100 mL of water and extracted with ethyl acetate (100 mL × 3). The combined extracts were washed with saturated NaCl solution (100 mL × 3), dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and finally purified by column chromatography to obtain compound Fmoc-Lys(Rho)-OtBu (525 mg, yield 19%).

[0045] 4. Compound Fmoc-Lys(Rho)-OtBu (525 mg, 0.617 mmol) was added to a round-bottom flask, followed by the addition of 5 mL LDCM and 5 mL TFA. The mixture was allowed to react overnight at room temperature. After the reaction was complete, the TFA was removed by rotary evaporation to obtain the target product Fmoc-Lys(Rho)-OH (470 mg).

[0046] Comparative Example: Synthesis of Rhodamine B-modified SUMO using a side-chain condensation strategy (1-47)

[0047] Weigh 312.65 mg (0.1 mmol) of 2-Cl-Trt-Cl resin with a degree of substitution of 0.32 mmol / g and place it in a polypeptide solid-phase synthesis tube. Add 10 mL of a 1:1 DMF / DCM mixture to the resin, allow it to swell for 20 minutes, and then use a diaphragm pump as a power source to dry the swollen product to obtain the swollen resin. Add 4 mL of 5% (v / v) hydrazine hydrate solution (0.2 mL hydrazine hydrate, 3.8 mL DMF) to the above resin, place it in a shaker at room temperature and shake for 30 minutes, then wash five times with DMF, add another 4 mL of 5% (v / v) hydrazine hydrate solution and react for 30 minutes, wash with the same washing method, add 4 mL of 5% (v / v) methanol solution (0.2 mL methanol, 3.8 mL DMF) and continue shaking for 10 minutes to block the unreacted active groups on the resin. After thorough washing with DMF, obtain the hydrazine resin.

[0048] Subsequent peptide synthesis steps were all performed using a CS Bio CS136 peptide synthesizer. Each standard amino acid coupling step mainly included: 1) adding a DMF solution containing 20% ​​(v / v) piperidine, reacting at room temperature for 5 minutes, washing three times with DMF, adding another 20% piperidine, reacting at room temperature for 10 minutes to completely remove the Fmoc protecting group of the amino group on the resin, and then washing the resin three times with DMF to remove residual piperidine; 2) adding 5 equivalents of amino acid (0.2M DMF stock solution), 5 equivalents of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 0.5M DMF stock solution), and 5 equivalents of N-hydroxy-7-azabenzotriazole (HOAt, 0.5M DMF stock solution). 1) Add 5 equivalents of amino acids (0.2M DMF stock solution), 5 equivalents of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 0.5M DMF stock solution), 5 equivalents of N-hydroxy-7-azabenzotriazole (HOAt, 0.5M DMF stock solution), and 10 equivalents of N,N-diisopropylethylamine (DIEA, 1.0M DMF stock solution). After reacting at room temperature for 30 minutes, wash three times with DMF. Using the above synthetic steps, the Fmoc-protected amino acids were sequentially and automatically coupled. The last Cys was Boc-Cys(Trt)-OH (peptide sequence Cys-AEEA2-K(Mtt)-AEEA2-Nle-ADEKPKEGVKTENNDHINLKVAGQDGSVVQFKIKRHTPLSKLMKAY), yielding Cys-AEEA2-K(Mtt)-AEEA2-SUMO (1-47)-CONHNH resin. After coupling, 4 mL of a mixed solution of hexafluoroisopropanol (HFIP) / 1,2-dichloroethane (DCE) containing 1-hydroxybenzotriazole (HOBt, 68 mg, 0.5 mmol, 5 eq) (volume ratio 1:1) was added to remove the Mtt protecting group. After shaking at room temperature for 3 minutes, the mixture was washed three times with DCM. The above steps were repeated six times with 4 mL of the mixed solution to completely remove the Mtt protecting group of lysine.

[0049] Take a small amount of the above resin and add 1 mL of a pre-prepared cleavage reagent (a mixture of trifluoroacetic acid, phenol, water, and triisopropylsilane, in a volume ratio of trifluoroacetic acid:water:phenol:triisopropylsilane = 88:5:5:2). React at room temperature for 1.5 hours. After cleaving the polypeptide chain from the resin, collect the filtrate into a centrifuge tube and concentrate the cleavage solution using nitrogen bubbling. Finally, when the cleavage solution is concentrated to less than 0.2 mL, add 1 mL of ice-cold diethyl ether for precipitation. Centrifuge at low speed (4500 rpm) to allow the crude peptide to settle to the bottom. Remove the supernatant and add ice-cold diethyl ether again. Sonicate until a crude peptide suspension is formed. Small molecule impurities that have been cleaved dissolve in the ice-cold diethyl ether and are then removed by centrifugation. After both processes, place the solid precipitate in a cool place to air dry, obtaining solid crude peptide. Take a small amount of crude peptide, dissolve it in an aqueous solution containing 20% ​​acetonitrile, filter it through a membrane, and analyze it using reversed-phase high-performance liquid chromatography (RP-HPLC). The acetonitrile concentration gradient was 20%-90% for 30 min. Chromatographic analysis followed by ESI-MS identification of the main peak verified the correctness of the crude peptide Cys-AEEA2-K-AEEA2-SUMO(1-47)-CONHNH2.

[0050] Rhodamine B (96 mg, 0.2 mmol, 2 eq), PyAOP (104 mg, 0.2 mmol, 2 eq), and HOAt (27 mg, 0.2 mmol, 2 eq) were then dissolved in 4 mL of DMF (Rhodamine B has low solubility and can be dissolved with oscillation and sonication). After activation for 1 minute, NMM (44 μL, 0.4 mmol, 4 eq) was added to the above hydrazine resin and placed in a shaker at room temperature for 12 hours to couple Rhodamine B with the ε-amino group of lysine residues. Finally, the Fmoc protecting group at the N-terminus of the peptide chain was removed. After washing the resin three times with DCM, the residual DCM solvent was removed by vacuum drying. Then, 5 mL of a pre-prepared cleavage reagent (a mixture of trifluoroacetic acid, phenol, water, and triisopropylsilane, in a volume ratio of 88:5:5:2) was added to the dried resin. The reaction was carried out at room temperature for 2.5 hours. The polypeptide chains were cleaved from the resin, and the filtrate was collected into centrifuge tubes. The cleavage solution was concentrated using nitrogen bubbling. Finally, when the cleavage solution was concentrated to less than 1 mL, 20 mL of ice-cold diethyl ether was added for precipitation. The mixture was centrifuged at low speed (4500 rpm) to allow the crude peptide to settle to the bottom. After removing the supernatant, ice-cold diethyl ether was added again, and the mixture was sonicated until a crude peptide suspension was formed. Small molecule impurities were dissolved in the ice-cold diethyl ether and removed by centrifugation. After both processes, the solid precipitate was air-dried in a cool place to obtain solid crude peptide. A small amount of crude peptide was dissolved in an aqueous solution containing 20% ​​acetonitrile, filtered through a membrane, and analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC). The analyte concentration gradient was 20%-90% acetonitrile, and the analysis time was 30 min. ESI-MS analysis after chromatographic analysis indicated that the main peak was from the unconverted rhodamine B-modified starting material (Cys-AEEA2-K-AEEA2-SUMO(1-47)-CONHNH2), while the rhodamine B-modified product (Cys-AEEA2-K(Rho)-AEEA2-SUMO(1-47)-CONHNH2) only accounted for 5% of the main peak area. Therefore, Cys-AEEA2-K(Rho)-AEEA2-SUMO(1-47)-CONHNH2 is difficult to obtain via lysine side-chain condensation.

[0051] Example 2: Synthesis of Rhodamine B-modified SUMO(1-47) using Fmoc-Lys(Rho)-OH

[0052] In this embodiment, Cys-AEEA2-K(Rho)-AEEA2-SUMO (1-47)-CONHNH2 was directly synthesized using Fmoc-Lys(Rho)-OH. The synthesis steps are similar to those in the comparative example, except that Fmoc-Lys(Mtt)-OH is replaced with Fmoc-Lys(Rho)-OH. After coupling, the steps for cutting the crude peptide from the resin and obtaining the solid crude peptide are the same as in the fourth section of the comparative example. A small amount of crude peptide was dissolved in an aqueous solution containing 20% ​​acetonitrile, filtered through a membrane, and analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC). The analyte gradient was 20%-90% acetonitrile concentration, and the analysis time was 30 min. After chromatographic analysis, the main peak was identified by ESI-MS, which confirmed that the main peak was the target product Cys-AEEA2-K(Rho)-AEEA2-SUMO (1-47)-CONHNH2. Therefore, Fmoc-Lys(Rho)-OH can be used to efficiently and rapidly obtain Rhodamine B-modified peptides that are difficult to obtain by lysine side chain condensation methods.

[0053] We infer that the full-length SUMO(1-47) generates a rigid secondary structure on the resin, resulting in the lysine side chain amino group at the modification site being embedded in the peptide chain. Therefore, in the comparative example, the synthesis of Rhodamine B-modified SUMO(1-47) via a side-chain condensation strategy was extremely low. Using the molecular building block Fmoc-Lys(Rho)-OH of this invention, condensation can occur during main chain extension, effectively avoiding this problem.

[0054] Example 3:

[0055] 263.15 mg (0.1 mmol) of Rink amide AM resin with a degree of substitution of 0.38 mmol / g was weighed and placed into a CS Bio CS136 peptide synthesizer. Each amino acid coupling step was the same as in the comparative example. Fmoc-Gly-OH, Fmoc-Glu(OAllyl)-OH, Nα-Fmoc-Nω-(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl)-D-arginine (Fmoc-D-Arg(Pbf)-OH), Nα-Fmoc-Nω-(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl)-L-arginine (Fmoc-Arg(Pbf)-OH), Fmoc-D-Arg(Pbf)-OH, and Fmoc-Arg(Pbf)-O were added sequentially. H, Fmoc-D-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Mtt)-OH are condensed into the resin to finally obtain linear R10 resin: Fmoc-K(Mtt)-RrRrRrRr-E(OAllyl)-G-CONH resin (R represents L-type Arg, r represents D-type Arg).

[0056] Add 4 mL of a mixed solution of Pd(PPh3)4 and PhSiH3 to the above resin to remove the Allyl protecting group from the glutamic acid side chain. React in a shaker at room temperature for 3 hours. Then wash the resin three times each with DMF, DCM, DMF, and DCM sequentially. The mixed solution is formulated as follows: Weigh Pd(PPh3)4 (115 mg, 0.1 mmol, 1 eq) and PhSiH3 (124 μL, 1 mmol, 10 eq) and add them to a 10 mL ep tube. Make up to 4 mL with DCM. Since there is excess palladium reagent remaining in the resin, use a DMF solution of palladium washing reagent (sodium diethyldithiocarbamate trihydrate) and react with shaking at room temperature for 5 minutes. Repeat the process until the resin is washed clean and the black color returns to a pale yellow. Subsequently, 4 mL of a 1:1 mixture of hexafluoroisopropanol (HFIP) and 1,2-dichloroethane (DCE) containing 1-hydroxybenzotriazole (HOBt, 68 mg, 0.5 mmol, 5 eq) was added to remove the Mtt protecting group. After shaking at room temperature for 3 minutes, the mixture was washed three times with DCM. This process was repeated six times with 4 mL of the mixture to completely remove the Mtt protecting group from lysine. Next, 3 mL of cyclizing reagent was added to the resin, and the synthesis tube was placed in a shaker at 37°C overnight to allow intramolecular cyclization of the exposed amino and carboxyl groups at both ends of the linear peptide molecule. The cyclizing reagent formulation was as follows: PyAOP (209 mg, 0.4 mmol, 4 eq), HOAt (55 mg, 0.4 mmol, 4 eq), and NMM (90 μL, 0.8 mmol, 8 eq), diluted to 3 mL with DMF. Next, using the CS Bio CS136 peptide synthesizer, following the same coupling steps, 2-[2-(N-Fmoc-amino)ethoxy]ethoxy]acetic acid (Fmoc-AEEA-OH), Fmoc-AEEA-OH, and Fmoc-Lys(Rho)-OH were sequentially condensed into the resin, and the last Fmoc was removed to obtain H2N-K(Rho)-AEEA2-cR10-G-CONH resin.

[0057] Add 5 mL of a pre-prepared cleavage reagent (a mixture of trifluoroacetic acid, phenol, water, and triisopropylsilane, in a volume ratio of 88:5:5:2) to the obtained resin. React at room temperature for 2.5 hours. After cleaving the polypeptide chain from the resin, collect the filtrate into a centrifuge tube and concentrate the cleavage solution using nitrogen bubbling. Finally, when the cleavage solution is concentrated to less than 1 mL, add 20 mL of ice-cold diethyl ether for precipitation. Centrifuge at low speed (4500 rpm) to allow the crude peptide to settle to the bottom. Remove the supernatant and add ice-cold diethyl ether again. Sonicate until a crude peptide suspension is formed. Small molecule impurities are dissolved in the ice-cold diethyl ether and removed by centrifugation. After both processes, air-dry the solid precipitate in a cool place to obtain solid crude peptide. Dissolve a small amount of crude peptide in an aqueous solution containing 5% acetonitrile, filter through a membrane, and analyze using reversed-phase high-performance liquid chromatography (RP-HPLC). The acetonitrile concentration gradient was 5%-70% for 30 min. Chromatographic analysis followed by ESI-MS identification of the main peak confirmed that it was the target product K(Rho)-AEEA2-cR10-G-CONH2.

[0058] Next, we used fluorescence live-cell imaging to detect the cell penetration of K(Rho)-AEEA2-cR10-G-CONH2 in HeLa cells. The lyophilized K(Rho)-AEEA2-cR10-G-CONH2 was directly dissolved in pure water to prepare a 500 μM stock solution. This solution was then added to the culture medium and incubated with the cells for 4 hours. After removing the remaining K(Rho)-AEEA2-cR10-G-CONH2, confocal microscopy revealed uniformly distributed red fluorescence within the cells, indicating that K(Rho)-AEEA2-cR10-G-CONH2 can efficiently penetrate the cell membrane and enter the cell.

Claims

1. A rhodamine B-modified lysine derivative, abbreviated as Fmoc-Lys(Rho)-OH, characterized in that... Its structural formula is shown below: 。 2. The method for synthesizing the rhodamine B-modified lysine derivative according to claim 1, characterized in that: First, Fmoc-Lys(Boc)-OtBu was synthesized from Fmoc-Lys(Boc)-OH. Then, the Boc protecting group was selectively removed, and it reacted with Rhodamine B to generate Fmoc-Lys(Rho)-OtBu. Finally, the tBu protecting group was removed to obtain Fmoc-Lys(Rho)-OH. The synthetic route is shown below: 。 3. The synthesis method according to claim 2, characterized in that... Includes the following steps: Step 1: Synthesis of Fmoc-Lys(Boc)-OtBu Fmoc-Lys(Boc)-OH was dissolved in dichloromethane, and then dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added to the reaction system in sequence and stirred until dissolved. Finally, tert-butanol was added and the reaction was carried out for 8-12 hours. After the reaction was completed, the precipitate was removed by filtration, and the resulting organic phase was concentrated under reduced pressure and purified by column chromatography to obtain Fmoc-Lys(Boc)-OtBu. Step 2: Synthesis of Fmoc-Lys(Rho)-OtBu 2a. Dissolve Fmoc-Lys(Boc)-OtBu in a 1,4-dioxane solution of hydrogen chloride and stir for 20 minutes. After the reaction was completed, the reaction solution was concentrated to obtain the crude product Fmoc-Lys-OtBu; 2b. Rhodamine B and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate were dissolved in N,N-dimethylformamide, and then N,N-diisopropylethylamine was added and the mixture was shaken and activated for 1 minute. The mixture was then added to a DMF solution containing the crude product Fmoc-Lys-OtBu and stirred for 2 hours. After the reaction was completed, the reaction system was diluted with water and extracted multiple times with ethyl acetate. The organic phase was collected, washed with saturated NaCl solution, dried, concentrated, and finally purified by column chromatography to obtain Fmoc-Lys(Rho)-OtBu. Step 3: Synthesis of Fmoc-Lys(Rho)-OH Fmoc-Lys(Rho)-OtBu was dissolved in DCM, and then trifluoroacetic acid was added and stirred overnight. After the reaction was completed, the remaining trifluoroacetic acid in the reaction system was removed by vacuum distillation to finally obtain Fmoc-Lys(Rho)-OH.

4. The synthesis method according to claim 3, characterized in that: In step 1, the molar ratio of Fmoc-Lys(Boc)-OH, tert-butanol, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1:2:2:0.

125.

5. The synthesis method according to claim 3, characterized in that: In step 2a, the concentration of hydrogen chloride in the 1,4-dioxane solution of hydrogen chloride is 4M.

6. The synthesis method according to claim 3, characterized in that: In step 2b, the molar ratio of crude product Fmoc-Lys-OtBu, Rhodamine B, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine is 1:1.1:1.1:

2.

7. The synthesis method according to claim 3, characterized in that: In step 3, the molar ratio of Fmoc-Lys(Rho)-OtBu to trifluoroacetic acid is 1:

60.

8. The synthesis method according to claim 3, characterized in that: In step 3, the volume ratio of DCM to trifluoroacetic acid is 1:

1.

9. The application of the rhodamine B-modified lysine derivative according to claim 1, characterized in that: By utilizing Fmoc-Lys(Rho)-OH and directly introducing it into the peptide sequence through an automated peptide solid-phase synthesizer, the rapid and automated acquisition of Rhodamine B-modified peptides can be achieved.

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