Preparation process of high-purity SMCC

Abstract

The application discloses a preparation process of high-purity SMCC and belongs to the technical field of organic synthesis. S1, siRNA is uniformly mixed with PBS buffer solution and cooled; S2, under ice bath condition, an NHS Ester DMSO solution is slowly and batchwise added dropwise; S3, after the dropwise addition is completed, the ice bath is removed, and the temperature is naturally increased to room temperature; a saturated sodium borate solution is batchwise added to adjust the pH value; and during the period, the reaction end point is monitored by using LC-MS; S4, after LC-MS detection, alcohol precipitation is carried out; after precipitation, the PBS buffer solution is used for dissolving and standby; S5, HPLC purification is carried out, SMCC NHS ester and by-reaction impurities are removed, alcohol precipitation is extracted dry, and high-purity SMCC finished products are obtained. The process provided by the application is green and safe, is suitable for industrialization, improves the raw material utilization rate through ice bath temperature control and batchwise dropwise addition, replaces benzene solvents with DMSO, reduces the toxicity risk, and omits a complex recrystallization step, thereby shortening the cycle.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology, specifically relating to a preparation process for high-purity SMCC. Background Technology

[0002] SMCC (4-(N-maleimide methyl)cyclohexane-1-carboxylic acid succinimide ester), as a heterobifunctional crosslinking agent, plays an irreplaceable role in bioconjugation chemistry and drug development. Its molecular structure contains two highly reactive groups: N-hydroxysuccinimide ester specifically binds to amino groups, while the maleimide group selectively reacts with thiol groups. This unique design allows SMCC to efficiently bridge different biomolecules, particularly in the preparation of antibody-drug conjugates, enabling the directional linking of toxin molecules to antibodies. The cyclohexane structure of SMCC is a key feature, effectively blocking steric interference between the two reactive groups and significantly reducing the hydrolysis rate of the maleimide group, allowing the modified protein to be lyophilized and stored, greatly improving storage stability. In applications, SMCC-activated phycocyanin and other reagents play a crucial role in high-end biodetection technologies such as flow cytometry, fluorescence immunoassay, and in vivo imaging. Its performance advantages are reflected in its extremely high quantum yield, photostability, and detection sensitivity that is hundreds of times higher than that of conventional organic fluorophores.

[0003] The purity requirements for SMCC are extremely stringent in biological applications, as trace impurities can trigger non-specific cross-linking, leading to antibody aggregation or decreased fluorescent labeling efficiency. Patent application CN 109678779 A discloses a method for purifying SMCC. This method involves recrystallizing crude SMCC containing impurities shown in Formula II obtained through chemical synthesis using acetonitrile as a solvent, effectively reducing the content of these impurities while improving SMCC purity. However, high yield losses in industrial production directly increase costs, impacting economic viability.

[0004] In existing SMCC preparation processes, the reaction is typically carried out in a saturated sodium borate or sodium carbonate / sodium bicarbonate buffer system, using a single-feed method. This technology has significant drawbacks: (1) pH control imbalance: The pH value of the buffer system is too high, which triggers a variety of side reactions, resulting in an increase in product impurities and a significant increase in the difficulty of subsequent purification; (2) Reaction exothermic runaway: The condensation reaction is exothermic, and the temperature rises sharply when the feed is added at once, which further accelerates side reactions such as hydrolysis and produces characteristic impurities with a molecular weight increase of 16 Da; (3) Product instability: SMCC is sensitive to hydrolysis, and traditional processes are difficult to inhibit its degradation, resulting in high impurity content and decreased yield in the final product.

[0005] As drug delivery systems evolve towards intelligence and precision, the demand for high-purity SMCC is becoming increasingly urgent. However, the impurity control deficiencies of existing processes severely limit their application potential in high-end fields such as oncology treatment, necessitating the development of mild and controllable new processes to overcome the bottlenecks in large-scale production and clinical translation. Summary of the Invention

[0006] The purpose of this invention is to provide a preparation process for high-purity SMCC, so as to improve the purity of the prepared SMCC.

[0007] The objective of this invention can be achieved through the following technical solutions: A process for preparing high-purity SMCC includes the following steps: S1. Mix siRNA with PBS buffer until homogeneous and cool. S2. Add NHS Ester DMSO solution slowly in batches under ice bath conditions; S3. After the addition is complete, remove the ice bath and allow it to rise naturally to room temperature. Add saturated sodium borate solution in batches to adjust the pH value. During this process, monitor the reaction endpoint with LC-MS. After passing the S4 and LC-MS tests, the sample was precipitated with alcohol, and then dissolved in PBS buffer for later use. S5. Perform HPLC purification to remove SMCC NHS ester and side reaction impurities, precipitate with alcohol and dry to obtain high-purity SMCC product.

[0008] In step S1, siRNA is thoroughly mixed with PBS buffer and cooled. The key is to maintain the stability of the siRNA's secondary structure. The PBS buffer provides an isotonic environment, preventing phosphodiester bond hydrolysis; low temperature control (below the siRNA melting temperature Tm) inhibits heat-induced denaturation and prevents single-strand dissociation. If the temperature is too high, the phosphodiester bond hydrolysis rate increases significantly, leading to siRNA degradation and affecting subsequent coupling efficiency.

[0009] In S2, the NHS Ester DMSO solution is slowly added dropwise in batches under ice bath conditions. The key challenge is to address the localized overheating issue caused by the exothermic reaction. The reaction between NHS Ester and the 5'-amino group is exothermic; adding it in one step would cause a sudden temperature rise, accelerating the hydrolysis of the phosphodiester bond in siRNA and the basic quenching of the NHS group (shortening the half-life). The batch-by-batch addition allows for the phased release of heat, and combined with stirring, ensures temperature uniformity, protecting the siRNA structure and preventing the loss of NHS activity. DMSO, as a highly polar solvent, enhances the solubility of NHS Ester, but the dropping rate must be controlled to prevent excessively high local concentrations that could trigger side reactions.

[0010] After removing the ice bath from reaction S3, the mixture was allowed to rise naturally to room temperature, and saturated sodium borate solution was added in batches to adjust the pH. The endpoint was monitored using LC-MS to precisely control the reaction process. Sodium borate provides a weakly alkaline environment to promote amino coupling, but adding it in batches avoids a sudden pH rise that could cause ring-opening of SMCC maleimide. LC-MS real-time monitoring of the disappearance of SS chain molecular weight serves as an endpoint marker to ensure complete reaction; if residual SS chains remain, NHS Ester can be added to salvage the reaction. This design balances reaction rate and impurity control, with a slightly acidic transition (subsequent pH adjustment) inhibiting maleimide hydrolysis.

[0011] After passing LC-MS analysis in step S4, purification was performed using alcohol precipitation. Unreacted SMCC was separated by utilizing its reduced solubility in alcohol solvents. The precipitate was then reconstituted with 0.01M PBS buffer to maintain a slightly acidic environment (pH 6-7) to prevent maleimide ring-opening. The PBS concentration was optimized to a low molar concentration to avoid interference from ionic strength in subsequent HPLC and to provide a stable buffer. Omitting this step would result in residual DMSO or salts affecting purification efficiency and leading to a decrease in the purity of the final product.

[0012] In S5, HPLC purification targets and removes SMCC NHS ester precursors and by-reaction impurities (such as hydrolysis products), achieving highly selective separation based on differences in molecular polarity and size. A C18 reversed-phase column can be used as the stationary phase, and gradient elution of the mobile phase precisely cuts off the target peak, ensuring a SMCC purity >98%. Relying on a single precipitation method cannot effectively separate structurally similar impurities; HPLC intervention is a crucial step in obtaining high purity, directly determining the bioactivity of the final product. SMCC is directly packaged and shipped after alcohol precipitation, significantly shortening the lyophilization time and preventing primer ring-opening.

[0013] Furthermore, the pH of the PBS buffer is 7.2-7.4.

[0014] Furthermore, the ratio of the saturated sodium borate solution to the reaction solution is (45-55) μL: 1000 μL.

[0015] PBS buffer is added to ensure the stability of the reaction system and prevent SMCC from ring-opening. To ensure complete reaction, saturated sodium borate solution is added in batches. MS analysis is performed midway through the reaction to ensure complete reaction. NHS Ester can be added again based on the MS results.

[0016] Furthermore, the structural formula of the NHS Ester is as follows: .

[0017] Furthermore, in the NHS Ester DMSO solution, the ratio of NHS Ester to DMSO is (0.8-1.2) g: 10 mL.

[0018] Furthermore, the siRNA is 5'-NH2-SiART101.

[0019] Furthermore, the ratio of siRNA, PBS buffer, NHS Ester DMSO solution and deionized water is (95-105) mg: 3 mL: (9-11) mL.

[0020] Furthermore, the cooling process involves placing the object in an ice bath to cool it to -4 to 4°C.

[0021] Furthermore, the ice bath conditions involve maintaining the temperature of the reaction system at -4 to 4°C using an ice bath.

[0022] The present invention employs ice bath temperature control (-4~4℃): maintaining the reaction system below the siRNA melting temperature (Tm) to protect the integrity of its secondary structure; and batch dropwise addition: controlling the phased release of reaction heat to avoid local overheating and ensure reaction uniformity.

[0023] Furthermore, the stepwise slow addition involves dividing the NHS Ester DMSO solution to be added into several equal portions, adding them slowly in batches while stirring.

[0024] The reaction between NHS Ester and the 5'-amino group of siRNA is an exothermic process. Traditional one-step addition can easily lead to a sudden increase in local temperature, causing the following problems: thermal denaturation of siRNA: the hydrolysis rate of phosphodiester bonds increases significantly at temperatures above 4°C; quenching of NHS activity: the half-life of the NHS group is shortened in an alkaline environment.

[0025] Furthermore, the monitoring reaction endpoint is when the molecular weight of the SS chain without SMCC disappears in the spectrum, indicating that the reaction is complete, while the molecular weight after SMCC is attached is 220 more.

[0026] The maleimide group of SMCC is prone to hydrolysis and ring-opening under alkaline conditions, generating inactive maleic amide. This hydrolysis reaction follows pseudo-first-order kinetics, and the rate constant k increases by 3 times when pH>7. This process is slightly acidic: using a NaH2PO4 buffer system, the pH is precisely controlled to be close to the stable range of maleimide (pH 6-7). The protonated environment inhibits nucleophilic attack by OH and reduces hydrolysis impurities.

[0027] The beneficial effects of this invention are: (1) The present invention adopts the strategy of ice bath temperature control and batch drop addition of NHSEsterDMSO solution to keep the reaction system below the melting temperature (Tm) of siRNA, protect its secondary structure integrity, and control the phased release of reaction heat to avoid local overheating and ensure reaction uniformity. The reaction endpoint (marked by the disappearance of SS chain molecular weight) is monitored in real time by LC-MS, replacing the traditional experience judgment, which shortens the reaction time, improves the utilization rate of raw materials, and effectively reduces the occurrence of side reactions.

[0028] (2) The process provided by the present invention is green and safe and suitable for industrialization. DMSO replaces benzene solvents, which significantly reduces the risk of toxicity. DMSO has better solubility for siRNA and NHSEster. The complex recrystallization step is omitted and HPLC is used for one-step purification, which shortens the production cycle, reduces yield loss, accurately removes SMCCNHS esters and hydrolysis impurities, and greatly improves product purity.

[0029] (3) In this invention, the pH of the system is maintained in the maleimide stable range of 6-7 by PBS buffer, and the temperature is controlled by ice bath to form a protonated environment to inhibit OH. - Nucleophilic attack reduces the hydrolysis rate of SMCC and improves product storage stability; the synergistic effect of various technical solutions achieves simultaneous optimization of purity, yield and safety, solving multiple technical pain points of existing processes. Detailed Implementation

[0030] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0031] General experimental materials siRNA: 5'-NH2-SiART101 (purity 99%); PBS buffer: 10×PBS Buffer, pH 7.4 NHS Ester: 98% purity; DMSO: analytical grade; Saturated sodium borate solution: analytical grade; Alcohol: Anhydrous ethanol (analytical grade); Instruments: LC-MS, HPLC.

[0032] Example 1 This embodiment provides a preparation process for high-purity SMCC, which is prepared through the following steps: S1. Mix 95mg of 5'-NH2-SiART101 with 3mL of PBS buffer and cool in an ice bath to -4℃. S2. Prepare NHS Ester DMSO solution (0.8g NHS Ester dissolved in 10mL DMSO). Under ice bath temperature control (-4℃), divide 9mL of this solution into 3 equal portions and slowly add them dropwise to the above system in batches, with an interval of 10min between each batch, while stirring. S3. After the addition is complete, remove the ice bath and allow it to rise naturally to room temperature. Add the saturated sodium borate solution in 4 batches at a ratio of 45 μL: 1000 μL (saturated sodium borate solution: reaction solution), with an interval of 10 min between each batch. During this period, monitor the reaction endpoint with LC-MS. Stop the reaction when the molecular weight of the SS chain disappears in the spectrum. After passing the S4 and LC-MS tests, the sample was precipitated with alcohol, and then dissolved in 10×PBS buffer for later use. S5. Perform HPLC purification to remove SMCCNHS ester and side reaction impurities, precipitate with alcohol and dry to obtain high-purity SMCC product.

[0033] Example 2 The difference between this embodiment and Embodiment 1 is that: The dosage of siRNA is 100 mg; The ratio of NHS Ester to DMSO in the NHS Ester DMSO solution is 1.0 g: 10 mL, and the solution volume is 10 mL. The cooling temperature and ice bath temperature control temperature are 0℃. The ratio of saturated sodium borate solution to reaction solution is 45 μL: 1000 μL.

[0034] The remaining raw materials and preparation process are the same as in Example 1.

[0035] Example 3 The difference between this embodiment and Embodiment 1 is that: The dosage of siRNA is 100 mg; The ratio of NHS Ester to DMSO in the NHS Ester DMSO solution is 1.0 g: 10 mL, and the solution volume is 3 mL. The cooling temperature and ice bath temperature control temperature are 4℃. The ratio of saturated sodium borate solution to reaction solution is 45 μL: 1000 μL.

[0036] The remaining raw materials and preparation process are the same as in Example 1.

[0037] Example 4 The difference between this embodiment and Embodiment 1 is that: The dosage of siRNA is 100 mg; The volume of NHS Ester DMSO solution used is 10 mL; The cooling temperature and ice bath temperature control temperature are 0℃. The ratio of saturated sodium borate solution to reaction solution is 45 μL: 1000 μL.

[0038] The remaining raw materials and preparation process are the same as in Example 1.

[0039] Example 5 The difference between this embodiment and Embodiment 1 is that: The ratio of NHS Ester to DMSO in the NHS Ester DMSO solution is 1.0 g: 10 mL.

[0040] The remaining raw materials and preparation process are the same as in Example 1.

[0041] Example 6 The difference between this embodiment and Embodiment 1 is that: The ratio of saturated sodium borate solution to reaction solution is 45 μL: 1000 μL; The drops were added in four batches, with a 10-minute interval between each batch.

[0042] The remaining raw materials and preparation process are the same as in Example 1.

[0043] Comparative Example 1 The difference between this comparative example and Example 1 is that the reaction was carried out without ice bath temperature control, maintaining room temperature (25°C) throughout. The specific implementation steps are as follows: S1. Mix 100 mg of 5'-NH2-SiART101 with 3 mL of PBS buffer and let stand at room temperature. S2. Prepare NHS Ester DMSO solution (1.0 g NHS Ester dissolved in 10 mL DMSO). Add 10 mL of this solution at room temperature while stirring. S3. Add saturated sodium borate solution in batches (at a ratio of 45 μL: 1000 μL), react for 10 minutes, and send the solution to MS for detection in a timely manner. Based on the detection results, NHS Ester DMSO solution may be added as needed. S4. Subsequent steps are the same as in Example 1.

[0044] The remaining raw materials and preparation process are the same as in Example 1.

[0045] Comparative Example 2 The difference between this comparative example and Example 1 is that both the NHS Ester DMSO solution and the saturated sodium borate solution were added at once. The specific implementation steps are as follows: S1. Mix 100 mg of 5'-NH2-SiART101 with 3 mL of PBS buffer and cool in an ice bath to -4°C. S2. Prepare NHS Ester DMSO solution (1.0g NHS Ester dissolved in 10mL DMSO). Add 10mL of this solution at once while stirring. S3. After the addition is complete, remove the ice bath and allow it to rise naturally to room temperature. Then, add a saturated sodium borate solution (at a ratio of 45 μL: 1000 μL) all at once, and monitor the reaction endpoint with LC-MS during the process. S4. Subsequent steps are the same as in Example 1.

[0046] The remaining raw materials and preparation process are the same as in Example 1.

[0047] Comparative Example 3 The difference between this comparative example and Example 1 is that toluene is used instead of DMSO as the solvent. The specific implementation steps are as follows: S1. Mix 100 mg of 5'-NH2-SiART101 with 3 mL of PBS buffer and cool in an ice bath to -4°C. S2. Prepare NHS Ester toluene solution (1.0 g NHS Ester dissolved in 10 mL toluene), and slowly add 10 mL of the solution dropwise in 3 batches under ice bath conditions; S3. Subsequent steps are the same as in Example 1.

[0048] The remaining raw materials and preparation process are the same as in Example 1.

[0049] Comparative Example 4 The difference between this comparative example and Example 1 is that there is no real-time LC-MS monitoring; the reaction is stopped after 2 hours based on experience. The specific implementation steps are as follows: S1. Mix 100 mg of 5'-NH2-SiART101 with 3 mL of PBS buffer and cool in an ice bath to -4°C. S2. Prepare NHS Ester DMSO solution (1.0g NHS Ester dissolved in 10mL DMSO), and slowly add 10mL of the solution in 3 batches. S3. After the addition is complete, remove the ice bath and allow it to rise naturally to room temperature. Add the saturated sodium borate solution in 3 batches and stop the reaction after 10 minutes. S4. Subsequent steps are the same as in Example 1.

[0050] The remaining raw materials and preparation process are the same as in Example 1.

[0051] Performance testing Performance tests were conducted on the SMCC products obtained in the examples and comparative examples, based on the following criteria: 1. Purity tests (area normalization method) were performed on the SMCC products obtained in the various embodiments and comparative examples of this application according to the high performance liquid chromatography method in General Chapter 0512 of Part IV of the Pharmacopoeia of the People's Republic of China, Part 2020. 2. The relative peak areas of the main hydrolytic impurities ([M+H]+ = SMCC molecular weight + 18) were semi-quantitatively analyzed by LC-MS; 3. Calculate the yield based on the amount of raw materials used in synthesis and the actual output; 4. Storage stability test: Store the product at 4℃ for 30 days and calculate the purity reduction rate; 5. Solvent Residue Test: The amount of solvent residue was determined by gas chromatography. The results are shown in Table 1: Table 1

[0052] As shown in Table 1, the SMCC products prepared in Examples 1-6 of this invention all achieved a purity of over 99.2%, with hydrolysis impurities and SMCCNHS ester residues all below 0.15%, yields exceeding 65%, a purity decrease rate of less than 0.8% after 30 days, and low solvent residue (≤28ppm), demonstrating excellent overall performance. Example 2, serving as the baseline example, exhibited the best performance across all aspects, with a purity as high as 99.7%, a yield of 66.3%, and good storage stability.

[0053] In contrast, the products of Comparative Example 1 (lacking ice bath temperature control), Comparative Example 2 (lacking batch dropwise addition), Comparative Example 3 (toluene replacing DMSO), and Comparative Example 4 (lacking LC-MS monitoring) all had a purity of less than 96%, significantly increased impurity content, yields of only 51.9%-56.4%, poor storage stability, and excessive solvent residue (Comparative Example 3). This fully demonstrates the key role of the various technical innovations and synergistic effects of the present invention in improving product performance.

[0054] In summary, the high-purity SMCC preparation process provided by this invention effectively solves the technical problems of existing processes, such as low purity, numerous side reactions, large yield losses, high toxicity, and poor stability, through synergistic innovations including ice bath temperature control, batch-by-batch addition, DMSO as a green solvent, real-time LC-MS monitoring, and HPLC purification. This process is green, safe, and suitable for industrialization. The prepared SMCC product has high purity, low impurity content, stable yield, and good storage properties, fully meeting the stringent requirements of the biomedical field and possessing extremely high application value and market prospects.

[0055] The above-disclosed embodiments are merely a few specific examples of the present invention. However, the embodiments of the present invention are not limited thereto, and any variations that can be conceived by those skilled in the art should fall within the protection scope of the present invention.

Claims

1. A preparation process for high-purity SMCC, characterized in that, Includes the following steps: S1. Mix siRNA with PBS buffer until homogeneous and cool. S2. Add NHS Ester DMSO solution slowly in batches under ice bath conditions; S3. After the addition is complete, remove the ice bath and allow it to rise naturally to room temperature. Add saturated sodium borate solution in batches to adjust the pH value. During this process, monitor the reaction endpoint with LC-MS. After passing the S4 and LC-MS tests, the sample was precipitated with alcohol, and then dissolved in PBS buffer for later use. S5. Perform HPLC purification to remove SMCC NHS ester and side reaction impurities, precipitate with alcohol and dry to obtain high-purity SMCC product.

2. The preparation process of high-purity SMCC according to claim 1, characterized in that, The PBS buffer is a 10×PBS buffer; The pH is 7.2-7.

4.

3. The preparation process of high-purity SMCC according to claim 1, characterized in that, The ratio of the saturated sodium borate solution to the reaction solution is (45-55) μL: 1000 μL.

4. The preparation process of high-purity SMCC according to claim 1, characterized in that, In the NHS Ester DMSO solution, the ratio of NHS Ester to DMSO is (0.8-1.2) g: 10 mL.

5. The preparation process of high-purity SMCC according to claim 1, characterized in that, The ratio of siRNA, PBS buffer, NHS Ester DMSO solution and deionized water is (95-105) mg: 3 mL: (9-11) mL.

6. The preparation process of high-purity SMCC according to claim 1, characterized in that, The siRNA is 5'-NH2-SiART101.

7. The preparation process of high-purity SMCC according to claim 1, characterized in that, The cooling process involves placing the item in an ice bath and cooling it to -4 to 4°C.

8. The preparation process of high-purity SMCC according to claim 1, characterized in that, The ice bath conditions are to maintain the temperature of the reaction system at -4 to 4°C by controlling the temperature of the ice bath.

9. The preparation process of high-purity SMCC according to claim 1, characterized in that, The stepwise slow addition involves dividing the NHS Ester DMSO solution to be added into several equal portions, adding them slowly in batches while stirring.

10. The preparation process of high-purity SMCC according to claim 1, characterized in that, The endpoint of the monitored reaction is when the unattached SS chain and the disappearance of the SMCC molecular weight in the spectrum indicate that the reaction is complete.

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