A method for constructing and applying a leucine R group
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-12
- Publication Date
- 2026-08-14
AI Technical Summary
然而，将氨基酸R基团转化为作为碱基替代物的寡核苷酸仍在很大程度上未被探索
[0036] 1. The present invention discloses a method for constructing a leucine R group, revealing the previously unexplored realm of leucine R groups in nucleotides and providing a precise platform for elucidating complex interactions. These findings open promising avenues for oligonucleotide engineering by introducing leucine elements, providing better stability, internalization ability, and potential applications in oligonucleotide therapy.
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Figure CN122563969A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to a method for constructing and applying a leucine R group. Background Technology
[0002] Leucine has the most hydrophobic side chain among natural amino acids and plays a crucial role in protein structure and protein-protein interactions. Various leucine-related motifs, such as polyleucine, leucine zippers, and dileucine, possess different functions and have been extensively studied. Furthermore, quantitative analysis of hydrophobic effects is important for controlling protein function; therefore, leucine motifs have been incorporated into artificial systems for quantitative research. The fusion of peptide bonds with nucleotides has led to the discovery of efficient biomolecules, such as peptide nucleic acids. However, the conversion of amino acid R groups into oligonucleotides as base substitutes remains largely unexplored. Therefore, there is a need to design a method for constructing and applying leucine R groups. Summary of the Invention
[0003] To overcome the shortcomings of the existing technology, a method for constructing the leucine R group and its application are provided.
[0004] This invention is achieved through the following scheme:
[0005] A method for constructing a leucine R group, the method comprising the following steps:
[0006] Step 1: Prepare leucine analogues by reacting chlorosugar with alcohol;
[0007] Step 2: Prepare leucine and its analogue phosphonamide modules using the phosphonamide triester method;
[0008] The three-step process involves using solid-phase automated modular synthesis technology to introduce the leucine phosphinoamide module into the nucleic acid aptamer sequence and the oligonucleotide sequence.
[0009] In the first step, deoxynucleosides with side chains of isobutane, neopentane, and tetramethylsilane are prepared by reacting chlorosugar with alcohol.
[0010] The specific steps of the first step are as follows: at room temperature, add compounds 5, 6, 7, triethylamine, and 4-dimethylaminopyridine to a dichloromethane solution of compound 4, and stir at room temperature for 12 hours.
[0011] After the reaction was completed, purification and filtration yielded colorless oily compounds 8, 9, and 10;
[0012] Do not add sodium hydroxide to the methanol / water solution of compounds 8, 9, and 10, and stir at room temperature for 1 hour;
[0013] The compounds were purified to obtain compounds 1, 2, and 3.
[0014] The structural formula of compound 1 is:
[0015] The structural formula of compound 2 is:
[0016] The structural formula of compound 3 is:
[0017] The structural formula of compound 4 is:
[0018] The structural formula of compound 5 is:
[0020] The structural formula of compound 6 is:
[0021] The structural formula of compound 7 is:
[0022] The structural formula of compound 8 is:
[0023] The structural formula of compound 9 is:
[0024] The structural formula of compound 10 is: .
[0025] The specific steps of the second step are as follows: at room temperature, DMTr-Cl and DMAP are added to the pyridine solutions of compounds 1, 2 and 3 obtained in the first step, respectively, and stirred for 3 hours;
[0026] After purification, compounds 11, 12, and 13 were obtained, respectively.
[0027] The mixture was cooled to 0°C. Then, chlorocyanoethyl phosphoric acid was added dropwise to the above solution. The reaction was heated to room temperature and stirred for 2 hours, and purified to obtain the desired products 14, 15, and 16.
[0028] The structural formula of compound 1 is: The structural formula of compound 2 is: The structural formula of compound 3 is: The structural formula of compound 11 is: The structural formula of compound 12 is: The structural formula of compound 13 is: The structural formula of compound 14 is: The structural formula of compound 15 is:
[0029] The structural formula of compound 16 is: .
[0030] An application of a method for constructing a leucine R group involves the programmable synthesis of a series of chimeric oligonucleotides with leucine, dileucine, or polyleucine motifs using phosphonamides containing a leucine R group and its analogues.
[0031] The leucine R group can be integrated into oligonucleotides as a base substitute, which can be used to enhance the biological activity of aptamers and antisense oligonucleotides.
[0032] The oligonucleotide contains polyleucines that induce the formation of helical structures, and the oligonucleotide can be used in engineered functional aptamers and DNA.
[0033] Incorporation of the bisleucine motif can be used to enhance the internalization of antisense oligonucleotides within cells.
[0034] The programmable fusion of the oligonucleotide with the R group can provide a molecular tool for studying protein mechanisms and new methods.
[0035] The beneficial effects of this invention are as follows:
[0036] 1. The present invention discloses a method for constructing a leucine R group, revealing the previously unexplored realm of leucine R groups in nucleotides and providing a precise platform for elucidating complex interactions. These findings open promising avenues for oligonucleotide engineering by introducing leucine elements, providing better stability, internalization ability, and potential applications in oligonucleotide therapy.
[0037] 2. The leucine R group of this invention not only elucidates the hydrophobic effect of leucine in the DNA framework, but also expands its applications to enhance the biological activity of aptamers and antisense oligonucleotides. Attached Figure Description
[0038] Figure 1 This is a flowchart illustrating the preparation process of leucine analogues.
[0039] Figure 2 This is a flowchart illustrating the preparation process of the leucine phosphonamide module.
[0040] Figure 3 A schematic diagram showing the molecular dynamics simulation and characterization results of polyleucine-modified oligonucleotides;
[0041] Figure 4 A schematic diagram showing the targeting characterization results of polyleucine-modified aptamers;
[0042] Figure 5This is a schematic diagram showing the internalization and interference characterization results of polyleucine-modified antisense oligonucleotides. Detailed Implementation
[0043] The preferred embodiments of the present invention are further described below:
[0044] A method for constructing a leucine R group, the method comprising the following steps:
[0045] Step 1: Prepare leucine analogues by reacting chlorosugar with alcohol;
[0046] Step 2: Prepare leucine and its analogue phosphonamide modules using the phosphonamide triester method;
[0047] The three-step process involves using solid-phase automated modular synthesis technology to introduce the leucine phosphinoamide module into the nucleic acid aptamer sequence and the oligonucleotide sequence.
[0048] In the first step, deoxynucleosides with side chains of isobutane, neopentane, and tetramethylsilane are prepared by reacting chlorosugar with alcohol.
[0049] The specific steps of the first step are as follows: at room temperature, add compounds 5, 6, 7, triethylamine, and 4-dimethylaminopyridine to a dichloromethane solution of compound 4, and stir at room temperature for 12 hours.
[0050] After the reaction was completed, purification and filtration yielded colorless oily compounds 8, 9, and 10;
[0051] Do not add sodium hydroxide to the methanol / water solution of compounds 8, 9, and 10, and stir at room temperature for 1 hour;
[0052] The compounds were purified to obtain compounds 1, 2, and 3.
[0053] The structural formula of compound 1 is: The structural formula of compound 2 is: The structural formula of compound 3 is: The structural formula of compound 4 is: The structural formula of compound 5 is: The structural formula of compound 6 is: The structural formula of compound 7 is: The structural formula of compound 8 is:
[0054] The structural formula of compound 9 is:
[0055] The structural formula of compound 10 is: .
[0056] The specific steps of the second step are as follows: at room temperature, DMTr-Cl and DMAP are added to the pyridine solutions of compounds 1, 2 and 3 obtained in the first step, respectively, and stirred for 3 hours;
[0057] After purification, compounds 11, 12, and 13 were obtained, respectively.
[0058] The mixture was cooled to 0°C. Then, chlorocyanoethyl phosphoric acid was added dropwise to the above solution. The reaction was heated to room temperature and stirred for 2 hours, and purified to obtain the desired products 14, 15, and 16.
[0059] The structural formula of compound 1 is:
[0060] The structural formula of compound 2 is:
[0061] The structural formula of compound 3 is:
[0062] The structural formula of compound 11 is:
[0063] The structural formula of compound 12 is:
[0064] The structural formula of compound 13 is:
[0065] The structural formula of compound 14 is:
[0066] The structural formula of compound 15 is:
[0067] The structural formula of compound 16 is: .
[0068] An application of a method for constructing a leucine R group involves the programmable synthesis of a series of chimeric oligonucleotides with leucine, dileucine, or polyleucine motifs using phosphonamides containing a leucine R group and its analogues.
[0069] The leucine R group can be integrated into oligonucleotides as a base substitute, which can be used to enhance the biological activity of aptamers and antisense oligonucleotides.
[0070] The oligonucleotide contains polyleucines that induce the formation of helical structures, and the oligonucleotide can be used in engineered functional aptamers and DNA.
[0071] Incorporation of the bisleucine motif can be used to enhance the internalization of antisense oligonucleotides within cells.
[0072] The programmable fusion of the oligonucleotide with the R group can provide a molecular tool for studying protein mechanisms and new methods.
[0073] The present application will be further described below with reference to specific embodiments:
[0074] (1) Compounds 5, 6, and 7, along with triethylamine (Et3N) and 4-dimethylaminopyridine (DMAP), were added to a dichloromethane solution of compound 4 at room temperature, and the mixture was stirred for 12 hours. After the reaction was complete, the mixture was purified and filtered to obtain colorless oily compounds 8, 9, and 10. Sodium hydroxide was then added to methanol / water solutions of compounds 8, 9, and 10, and the mixture was stirred for 1 hour at room temperature. Purification yielded compounds 1, 2, and 3. The reaction process is shown in the attached figure. Figure 1 As shown.
[0075] (2) At room temperature, DMTr-Cl and DMAP were added to pyridine solutions of compounds 1, 2, and 3, respectively, and stirred for 3 hours. After purification, compounds 11, 12, and 13 were obtained, respectively. The mixtures were cooled to 0°C. Then, chlorocyanoethyl phosphoric acid was added dropwise to the above solutions. The reaction was heated to room temperature and stirred for 2 hours. After purification, the desired products 14, 15, and 16 were obtained. The reaction process is shown in the attached figure. Figure 2 As shown.
[0076] (3) Using solid-phase automated modular synthesis technology, the leucine phosphamide module was inserted into the oligonucleotide sequence using a DNA synthesizer. The preparation process is shown in the attached figure. Figure 3 As shown.
[0077] (4) To analyze the molecular dynamics simulations of polyleucine-modified oligonucleotides, the initial structure of the DNA used in the MD simulation was constructed using 3DNA (http: / / w3dna.rutgers.edu / rebuild / fiberch) based on the residue sequence. The “L” residues were plotted using ChemDraw software. Bonds were established between the “L” residues and the DNA structure using Pymol software. The molecular topology file for the “L” residues was provided by AutomatedTopology Builder and Repository version 3.0 (https: / / atb.uq.edu.au / ). MD simulations were performed using GROMACS 5.1.4 software combined with the GROMOS96 54A7 force field. Water molecules were represented using the SPC / E model. Finally, 22 Cl ions were randomly added to replace water molecules to neutralize the simulation system. The time step was set to 2 fs, with a cutoff of 1.2 nm for short-range van der Waals and electrostatics. Lennard-Jones interactions were directly truncated at 1.2 nm. Bonds were constrained using the LINCS algorithm. The system first achieved energy minimization over 5000 steps. After minimization, the system was gradually heated from 0K to 300K under standard constant volume conditions, then the pressure was balanced to 300K in the NPT ensemble, and a production simulation was performed at 300K and 1 bar for 600ns. Trajectory coordinates were saved every 100ps throughout the entire MD run.
[0078] To analyze the stability of the helix, fluorescence measurements were performed using an RF-5301-PC fluorescence spectrophotometer (Shimadzu, Japan). Excitation and emission wavelengths were 490 nm and 520 nm, respectively, with a bandwidth of 5 nm. Background fluorescence was monitored using 100 nM FNAs (200 μL of 20 mM Tris-HCl, pH 7.5). After obtaining a stable fluorescence signal, an excess of complementary DNA to the loop was added. Fluorescence intensity was recorded as signal fluorescence. Results are attached. Figure 3 As shown.
[0079] The α-helix formed by polyleucine residues within proteins plays a crucial role in protein structure and function. We hypothesized that polyleucine residues also promote helical structure formation within the DNA backbone. Therefore, molecular dynamics (MD) simulations were conducted to investigate this hypothesis. In this study, we designed the oligonucleotide Leu-6, whose initial structure (I in the rod model)... Figure 3 A) Constructed using 3DNA (http: / / web.x3dna.org / ). MD simulations confirmed that the oligonucleotide tends to form a thermodynamically stable hairpin structure with intramolecular double strands (II in the rod model). Figure 3A). Based on MD simulation results, oligonucleotides OMB- containing different types of hydrophobic groups were designed and synthesized.
[0080] Leu5, OMB-Leu6, OMB-CSi, and OMB-Si. All OMBs are labeled with a fluorescent group (FAM) at the 3' end and a quencher (Dabcyl) at the 5' end. When polyleucine residues (poly-CSi or poly-Si) in the DNA backbone aggregate to form an intramolecular double-stranded structure, the fluorescent group and quencher are close together, producing a weak fluorescence signal. However, adding sequence complementary oligonucleotides (cDNA) to the loop region into the buffer solution results in a significant increase in fluorescence by forming a rigid and more stable DNA double strand, which moves the FAM away from the Dabcyl. Figure 3 B). Therefore, fluorescence changes can reflect the structural information within the DNA backbone incorporating polyleucines; furthermore, the stability of the helical structure can be visually demonstrated through fluorescence. Figure 3 As shown, OMB- was detected.
[0081] The fluorescence intensities of Leu5, OMB-Leu6, OMB-CSi, and OMB-Si were used as background values (green line), and the fluorescence intensity after cDNA addition was used as the signal value (red line). The signal-to-background ratio (S / B) of OMB-Leu6 was approximately 3.52. Figure 3 C), almost the S / B ratio of OMB-Leu5 (1.68, Figure 3 Twice that of D). These fluorescence changes indicate that a helical structure has formed in the DNA backbone via polyleucine fragments. It is important to note that stability is related to the number of consecutive leucine units. For example, the oligonucleotide OMB-Leu6 has 6 consecutive leucine units, while OMB-Leu5 has 5. When there are fewer than 5 leucine units, an intramolecular helical structure may not form. Replacing the 6 consecutive leucine units in oligonucleotide OMB-Leu6 with similar CSi or Si yields oligonucleotides OMB-CSi or OMB-Si, respectively. Figure 3 As shown in E, the S / B ratio of OMB-CSi is 13.03, which is comparable to the S / B ratio of classic molecular beacons composed of natural bases. Interestingly, the S / B ratio of OMB-Si is 27.11. Figure 3 F), indicating that OMB-Si has the most stable double-chain structure among all analogues. The hydrophobicity of leucine (Leu), CSi, and Si gradually increases.
[0082] The stability of double-stranded natural oligonucleotides is generally characterized by their melting temperature, which can be precisely measured. However, thermodynamic studies are not applicable to helical double strands assembled from OMB-Leu6, OMB-CSi, and OMB-Si because hydrophobic interactions are inert to temperature changes. Therefore, to further evaluate the stability of the OMB-Leu6 and OMB-CSi helices, we performed quantitative analysis using a competition experiment. Molecular beacons OMB-Leu6 and OMB-CSi were mixed with sequence-complementary oligonucleotides (ranging from 9 to 19 mers), which could hybridize with sequences that form intermolecular helices. This is a competition between intermolecular helices formed by hydrogen bonds between AT and CG base pairs and intramolecular helices formed by hydrophobic aggregation between Leu or CSi. Fluorescence changes provide direct evidence of competition and equilibrium, both of which were detected separately. The addition of the shortest oligomer, the 9-mer, to OMB-Leu6 or OMB-CSi had a negligible effect on fluorescence, indicating that the strength of a 9-base DNA double strand is weaker than any hydrophobic helix. Figure 3 G and 3H). 11mer or any longer oligomer can effectively dissociate the intramolecular double chain of OMB-Leu6, while only the longest 19mer can fully open the hairpin structure of OMB-CSi.
[0083] Circular dichroism (CD) spectroscopy is highly sensitive to DNA structure, and hairpin structures were further characterized using this technique. At the same concentration and in the same buffer solution, OMB-Leu6 exhibited the lowest CD intensity, while OMB-Si showed the highest, indicating that hydrophobicity directly determines the stability of the double strand. Figure 3 I).
[0084] To analyze the in vitro targeting of polyleucine-modified aptamers, HCT116 cells were cultured at 1 × 10⁶ cells per well. 5 Cells were seeded at a density in confocal culture dishes with a glass bottom and incubated overnight. Cells were incubated with 500 nM aptamers at 4°C for 30 min, then incubated with FITC-labeled anti-ptk7 (R&D) antibody for 30 min, followed by nuclear staining at 37°C for 20 min. The prepared samples were studied using a confocal microscope (Leica TCS SP8). Figure 4 As shown in Figure A, both Sgc8c and Sgc8-Si6 (red) show obvious co-localization with PTK7 (green).
[0085] To analyze the in vivo targeting of the polyleucine-modified aptamer, 4-week-old female BALB / c nude mice (Shanghai Experimental Animal Co., Ltd.) were subcutaneously injected with 1×10 6HCT116 cells were injected into the right hind limb dorsal region of mice. After the mice were anesthetized, when the tumor size reached 300 mm³, Cy5-labeled SGC8C or SGC8C-Si6 (20 μM, 100 μL) was injected intravenously. Images were obtained at specific time points (0, 1, 3, 6, 9, 24, 36, and 48 h), as shown in the attached image. Figure 4 As shown. The results indicate that the addition of the hydrophobic duplex prolonged the cycle time ( Figure 4 B), which increased the enrichment of Sgc8c at the tumor site. Figure 4 C).
[0086] To investigate the internalization ability of polyleucine-modified antisense oligonucleotides, HCT116 cells were cultured at 1 × 10⁶ cells per well. 5 Cells were seeded at a density in confocal culture dishes with a glass bottom and incubated overnight. Cells were then incubated with 500 nM aptamers at 37°C for 30 min, followed by nuclear staining at 37°C for 20 min. The prepared samples were studied using a confocal microscope (Leica TCS SP8). Results are attached. Figure 5 As shown.
[0087] To investigate the interference ability of polyleucine-modified antisense oligonucleotides, HCT116 cells were seeded in 6-well plates with 5000 cells per well and cultured for 24 hours. ASO targeting STAT3, STAT3, or siRNA-Leu (200 nM) was transfected into cells containing 6 μL of Lipo2000 (Thermo), or incubated with cells (1000 nM) for 48 hours. RNA was extracted using the RNeasy kit (QIAGEN), and real-time PCR was performed using the HitScript II One Step qRT-PCR SYRB Green kit (Vazyme). CT values were measured using a z480 (Roche) microscope, and relative expression levels were calculated using 2^^CT. Results are attached. Figure 5 As shown.
[0088] like Figure 5 As shown in Figures A and B, the internalization of the STAT3-Leu group was improved compared to the STAT3 group, indicating that the dileucine group improved the internalization of the modified oligonucleotide. This verified the cellular uptake capacity of the dileucine group. To verify whether the introduction of the dileucine group affected the gene knockout effect, we performed real-time PCR detection. Figure 5 As shown in C, STAT3 and STAT3-Leu
[0089] Transfection also downregulated the expression of this gene in HCT116 cells, indicating that the modification had a negligible effect on the ASO gene knockout effect. Finally, the gene knockout efficiency of STAT3-Leu and control STAT3 was tested. Figure 5 As shown in D, STAT3-Leu exhibits better gene regulatory activity than STAT3 in untransfected cells, suggesting that programmable dileucine-oligonucleotide chimeras may be further applied.
[0090] This invention addresses the problem of synthesizing leucine R groups and integrating them as base substitutes into oligonucleotides, elucidates the hydrophobic effect of leucine within the DNA framework, and expands its applicability in enhancing the bioactivity of aptamers and antisense oligonucleotides.
[0091] Although the technical solutions of the present invention have been described and enumerated in detail, it should be understood that modifications to the above embodiments or the adoption of equivalent alternatives are obvious to those skilled in the art. Such modifications or improvements made without departing from the spirit of the present invention are all within the scope of protection claimed by the present invention.
Claims
1. A method for constructing a leucine R group, characterized in that, The method includes the following steps: Step 1: Prepare leucine analogues by reacting chlorosugar with alcohol; The second step involves preparing leucine and its analogue phosphamide modules using the phosphamide triester method. The third step involves using solid-phase automated modular synthesis technology to introduce the leucine phosphinoamide module into the nucleic acid aptamer sequence and the oligonucleotide sequence.
2. The method for constructing a leucine R group according to claim 1, characterized in that: In the first step, deoxynucleosides with side chains of isobutane, neopentane, and tetramethylsilane are prepared by reacting chlorosugar with alcohol.
3. The method for constructing a leucine R group according to claim 2, characterized in that: The specific steps of the first step are as follows: at room temperature, add compounds 5, 6, 7, triethylamine, and 4-dimethylaminopyridine to a dichloromethane solution of compound 4, and stir at room temperature for 12 hours. After the reaction was completed, the mixture was purified and filtered to obtain colorless oily compounds 8, 9, and 10. Sodium hydroxide was added to methanol / water solutions of compounds 8, 9, and 10, respectively, and the mixtures were stirred at room temperature for 1 hour. The compounds were purified to obtain compounds 1, 2, and 3. The structural formula of compound 1 is: The structural formula of compound 2 is: The structural formula of compound 3 is: The structural formula of compound 4 is: The structural formula of compound 5 is: The structural formula of compound 6 is: The structural formula of compound 7 is: The structural formula of compound 8 is: The structural formula of compound 9 is: The structural formula of compound 10 is: .
4. The method for constructing a leucine R group according to claim 1, characterized in that: The specific steps of the second step are as follows: at room temperature, DMTr-Cl and DMAP are added to the pyridine solutions of compounds 1, 2 and 3 obtained in the first step, respectively, and stirred for 3 hours; After purification, compounds 11, 12, and 13 were obtained, respectively. The mixture was cooled to 0°C. Then, chlorocyanoethyl phosphoric acid was added dropwise to the above solution. The reaction was heated to room temperature and stirred for 2 hours, and purified to obtain the desired products 14, 15, and 16. The structural formula of compound 1 is: The structural formula of compound 2 is: The structural formula of compound 3 is: The structural formula of compound 11 is: The structural formula of compound 12 is: The structural formula of compound 13 is: The structural formula of compound 14 is: The structural formula of compound 15 is: The structural formula of compound 16 is: .
5. The application of a method for constructing the leucine R group as described in any one of claims 1-4, characterized in that: A series of chimeric oligonucleotides with leucine, dileucine, or polyleucine motifs were prepared by programmable synthesis using phosphonamides containing leucine R groups and their analogues.
6. The application of the method for constructing a leucine R group according to claim 5, characterized in that: Integrating the leucine R group as a base substitute into oligonucleotides can be used to enhance the biological activity of aptamers and antisense oligonucleotides.
7. The application of the method for constructing a leucine R group according to claim 6, characterized in that: The oligonucleotide contains polyleucines that induce the formation of helical structures, and the oligonucleotide is then used in engineered functional aptamers and DNA.
8. The application of the method for constructing the leucine R group according to claim 5, characterized in that: Incorporation of the bis-leucine motif is applied to enhance the internalization of antisense oligonucleotides within cells.
9. The application of the method for constructing a leucine R group according to claim 5, characterized in that: The programmable fusion of the oligonucleotide with the R group provides a new molecular tool for studying protein mechanisms.