Efficient and green synthesis method of 3 '-amino nucleotide

Abstract

The invention is suitable for the technical field of nucleotide synthesis, and provides a high-efficiency green synthesis method of 3 '-amino nucleotide, which is green and environment-friendly, and efficiently adapts to industrial production. Pure water is used as a unique solvent, so that no harmful waste is discharged; 5% of the Pd / C catalyst can be recycled, the low-concentration TEAB eluent can be recycled, and the green chemistry concept is met. The Pd / C catalyst has the advantages of high selectivity, few side reactions (impurities are less than or equal to 5%), mild reaction conditions (normal-pressure hydrogen and 50 DEG C), time consumption of only 2 hours and raw material conversion rate of more than or equal to 99%. DEAE resin and low-concentration gradient elution purification are adopted, the HPLC purity of the product is larger than or equal to 95% (part is larger than or equal to 98%), and the molar yield is 46-85% and is superior to that of an existing method; only two-step core operation is adopted, post-treatment is simple and convenient, corrosion to equipment is small, the method can adapt to 3 '-azido nucleotide raw materials with various substituent groups, and the preparation requirements of different drug intermediates are met.

Description

Technical Field

[0001] This invention relates to the field of nucleotide synthesis technology, specifically a highly efficient and green method for synthesizing 3'-amino nucleotides. Background Technology

[0002] Cancer and human immunodeficiency virus (HIV) infection are major diseases that seriously threaten human health and life worldwide. In the field of cancer treatment, oligonucleotide therapy, as a cutting-edge treatment strategy, inhibits abnormal gene activity by specifically binding to target genes or mRNA through artificially synthesized DNA or RNA sequences, providing a new direction for cancer prevention and treatment. Among them, 3'-aminonucleotides, as a novel dideoxynucleotide derivative, have shown great application potential in oligonucleotide drug design due to their superior water solubility and stability compared to traditional derivatives, and can significantly improve drug resistance to nucleases and target binding specificity.

[0003] In the field of HIV treatment, dideoxynucleotide analogs are commonly used and highly effective inhibitors in clinical practice. They inhibit viral replication by inserting into the viral DNA strand, causing strand termination, and blocking the reverse transcription process. 3'-Aminonucleotides, as a rising star in the dideoxynucleotide family, are considered important candidate substances for developing novel anti-HIV drugs due to their excellent physicochemical properties.

[0004] However, the preparation of 3'-aminonucleotides currently faces significant challenges. Traditional biosynthetic methods require the cultivation of specific expression enzymes, which not only imposes stringent requirements on synthetic equipment and production scale but also involves cumbersome and inefficient post-processing steps, making it difficult to meet the large-scale clinical demand for these compounds. Furthermore, existing synthetic methods lack green and environmentally friendly designs, failing to align with the sustainable development principles of modern pharmaceutical synthesis.

[0005] Therefore, developing an efficient and green chemical synthesis method for 3'-aminonucleotides, breaking through the bottlenecks of existing preparation technologies, and achieving low-cost, large-scale production is of great significance for promoting the research and application of novel anticancer and anti-HIV drugs, and has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of existing 3'-aminonucleotide synthesis methods, such as "high requirements for biological equipment, large pollution from chemical methods, many product impurities, and unstable yields," and provides a highly efficient and green synthesis method using water as a solvent and Pd / C catalytic hydrogen reduction to achieve high purity and stable yield of the target product, thus meeting the needs of industrial production.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A highly efficient and green method for the synthesis of 3'-amino nucleotides is disclosed. The core method uses 3'-azidonucleotides as raw materials and completes the synthesis through a two-step process of "catalytic reduction-cation exchange purification." Specific technical details are as follows:

[0009] The raw materials are 3'-azidonucleotides (in the general structural formula, R1 is H, OH, or SH, and R2 is a monophosphate, diphosphate, triphosphate, or polyphosphate), including but not limited to 3'-N3-dATP, 3'-N3-dCMP, 3'-N3-dGDP, 3'-N3-GDP, 3'-N3-dGQP, etc., which can be obtained from commercially available conventional reagents or through simple synthesis;

[0010] Pure water is used as the sole solvent, eliminating the need for any organic solvents. This method is environmentally friendly and can fully dissolve the raw materials, avoiding pollution and product separation problems caused by organic solvents.

[0011] The catalyst used is a 5% Pd / C catalyst, which has high catalytic activity and strong selectivity. It can specifically reduce azide groups (-N3) to amino groups (-NH2), effectively suppressing side reactions such as azide group desorption and ensuring product purity.

[0012] DEAE resin (diethylaminoethyl cation exchange resin) was selected as the purification medium and eluent. Its surface functional groups can form specific adsorption with 3'-amino nucleotides, resulting in excellent separation selectivity. TEAB (tetraethylammonium bromide) aqueous solution was used as the eluent. The low concentration gradient of 0.1mM to 0.3mM can precisely control the adsorption-desorption balance, achieving efficient separation of the target product from trace impurities, while avoiding the decrease in product purity caused by high concentrations of eluent.

[0013] Step S1:

[0014] Dissolve 1 mmol of 3'-azidonucleotide raw material in 10 mL of pure water at a ratio of 1:10 (mmol:mL) and stir until completely dissolved. Add 10 wt% of 5% Pd / C catalyst, purge with hydrogen gas at atmospheric pressure, control the reaction temperature at 50 °C, and stir for 2 h. Monitor the reaction progress in real time by HPLC, and terminate the reaction after the raw material peak has completely disappeared.

[0015] Step S2:

[0016] Diatomaceous earth was added to the reaction system as a filter aid, and 5% Pd / C catalyst was removed by filtration. The filtrate was collected. The filtrate was diluted with pure water to 1 L (concentration 1 mmol / L) and slowly passed into a pretreated DEAE resin column. Gradient elution was performed using 0.1 mM to 0.3 mM TEAB aqueous solution, with elution gradients of 0.1 mM (elution of 10 column volumes), 0.2 mM (elution of 10 column volumes), and 0.3 mM (elution of 10 column volumes). The eluent was detected by online HPLC, and the elution peak corresponding to the target product was collected. The target eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40 °C and freeze-dried to obtain a white solid 3' amino nucleotide.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] 1. This invention uses pure water as the sole solvent, eliminating the need for organic solvents and producing no harmful waste during the reaction process; the 5% Pd / C catalyst is recyclable and regenerable, and the low-concentration TEAB eluent is reusable, fully complying with the principles of green chemistry.

[0019] 2. The Pd / C catalyst of this invention exhibits high selectivity for azide reduction, effectively avoiding side reactions such as azide desorption, and the product impurity content is ≤5%; the reaction conditions are mild (atmospheric pressure hydrogen, 50℃), energy consumption is low, the reaction time is only 2 hours, and the feed conversion rate is ≥99%;

[0020] 3. This invention employs a purification method using DEAE resin + 0.1mM~0.3mM MTEAB low-concentration gradient elution, achieving a product HPLC purity ≥95%, with some products reaching purity above 98%. The molar yield is consistently between 46% and 85%, far superior to existing chemical synthesis methods (yields are generally below 50%). Only two core operations (reduction + purification) are required, eliminating the need for complex chromatographic equipment in post-processing. The low-concentration eluent has minimal corrosiveness to equipment, and the filtration, elution, concentration, and lyophilization processes are simple, making it suitable for large-scale industrial production. It is compatible with 3'-azidonucleotide raw materials with different R1 (H, OH, SH) and R2 (monophosphate, diphosphate, triphosphate, polyphosphate) substituents. DEAE resin exhibits strong compatibility in adsorption and elution of various nucleotides, meeting the preparation requirements of different pharmaceutical intermediates. Attached Figure Description

[0021] Figure 1 This is a synthetic route diagram of the 3' amino nucleotide of the present invention;

[0022] Figure 2 The HPLC spectrum of 3'-amino-dATP prepared in Example 1 of this invention;

[0023] Figure 3 The HPLC spectrum of 3'-amino-dCMP prepared in Example 2 of this invention;

[0024] Figure 4 The HPLC spectrum of 3'-amino-dGDP prepared in Example 3 of this invention;

[0025] Figure 5 The HPLC spectrum of 3'-amino-GDP prepared in Example 4 of this invention;

[0026] Figure 6 The HPLC spectrum of 3'-amino-dGQP prepared in Example 5 of this invention is shown. Detailed Implementation

[0027] The technical solution of this patent will be further described in detail below with reference to specific embodiments.

[0028] Example 1

[0029] Synthesis of 3'-amino-dATP:

[0030]

[0031] S1. Reduction reaction: Take 3'-N3-dATP (1 mmol, 491 mg), add 10 mL of ultrapure water, stir to dissolve; add 5% Pd / C catalyst (49.1 mg, 10 wt%), pass hydrogen gas at normal pressure, and stir at 50 °C for 2 h; HPLC detection showed that the starting material peak completely disappeared.

[0032] S2. Purification: Add 0.5g of diatomaceous earth to the reaction solution, stir well, and filter to remove the insoluble Pd / C catalyst; dilute the filtrate with ultrapure water to 1L and slowly inject it into the pretreated DEAE resin column; elute with a gradient of 0.1mM~0.3mM MTEAB aqueous solution at a flow rate of 1.5mL / min, and collect the target product peak (retention time 8.538min) by online HPLC detection; concentrate the target eluent to 100mL under reduced pressure at 40℃, freeze-dry, and obtain 295mg of off-white solid 3'-amino-dATP, with a molar yield of 60% and an HPLC purity of 97.59%.

[0033] Example 2

[0034] Synthesis of 3'-amino-dCMP:

[0035]

[0036] S1. Reduction reaction: Take 3'-N3-dCMP (1 mmol, 327 mg), add 10 mL of ultrapure water, and stir to dissolve; add 5% Pd / C catalyst (32.7 mg, 10 wt%), introduce hydrogen gas at normal pressure, and stir at 50 °C for 2 h; HPLC detection showed that the starting material peak completely disappeared.

[0037] S2. Purification: After the reaction solution was filtered through diatomaceous earth, the filtrate was diluted to 1L and injected into a DEAE resin column; gradient elution was performed with 0.1mM~0.3mM MTEAB aqueous solution, and the target product peak was collected by online HPLC detection; the solution was concentrated and freeze-dried to obtain 271mg of off-white solid 3'-amino-dCMP, with a molar yield of 83% and an HPLC purity of 98.13%.

[0038] Example 3

[0039] Synthesis of 3'-amino-dGDP:

[0040]

[0041] S1. Reduction reaction: Take 3'-N3-dGDP (1 mmol, 463 mg), add 10 mL of ultrapure water, stir to dissolve; add 5% Pd / C catalyst (46.3 mg, 10 wt%), pass hydrogen gas at normal pressure, and stir at 50 °C for 2 h; HPLC detection showed that the starting material peak completely disappeared.

[0042] S2. Purification: After the reaction solution was filtered through diatomaceous earth, the filtrate was diluted to 1L and injected into a DEAE resin column; gradient elution was performed with 0.1mM~0.3mM MTEAB aqueous solution, and the target product peak was collected by online HPLC detection; the solution was concentrated and freeze-dried to obtain 352mg of off-white solid 3'-amino-dGDP, with a molar yield of 76% and an HPLC purity of 97.67%.

[0043] Example 4

[0044] Synthesis of 3'-amino-GDP:

[0045]

[0046] S1. Reduction reaction: Take 3'-N3-GDP (1 mmol, 445 mg), add 10 mL of ultrapure water, stir to dissolve; add 5% Pd / C catalyst (44.5 mg, 10 wt%), pass hydrogen gas at normal pressure, and stir at 50 °C for 2 h; HPLC detection showed that the starting material peak completely disappeared.

[0047] S2. Purification: After the reaction solution was filtered through diatomaceous earth, the filtrate was diluted to 1L and injected into a DEAE resin column; gradient elution was performed with 0.1mM~0.3mM MTEAB aqueous solution, and the target product peak was collected by online HPLC detection; the solution was concentrated and freeze-dried to obtain 378mg of off-white solid 3'-amino-GDP, with a molar yield of 85% and an HPLC purity of 98.02%.

[0048] Example 5

[0049] Synthesis of 3'-amino-dGQP:

[0050]

[0051] S1. Reduction reaction: Take 3'-N3-dGQP (1 mmol, 512 mg), add 10 mL of ultrapure water, and stir to dissolve; add 5% Pd / C catalyst (51.2 mg, 10 wt%), introduce hydrogen gas at normal pressure, and stir at 50 °C for 2 h; HPLC detection showed that the starting material peak completely disappeared.

[0052] S2. Purification: After the reaction solution was filtered through diatomaceous earth, the filtrate was diluted to 1L and injected into a DEAE resin column; gradient elution was performed with 0.1mM~0.3mM MTEAB aqueous solution, and the target product peak was collected by online HPLC detection; the solution was concentrated and freeze-dried to obtain 235mg of off-white solid 3'-amino-dGQP, with a molar yield of 46% and an HPLC purity of 95.15%.

[0053] It is necessary to explain that in the above embodiments:

[0054] 1. A stable hydrogen atmosphere must be maintained during the reaction to prevent air from entering and causing catalyst deactivation, which would affect the reaction conversion rate;

[0055] 2. During the elution process, the concentration gradient and flow rate of the TEAB aqueous solution must be strictly controlled to avoid co-elution of the target product and impurities, which would lead to a decrease in purity.

[0056] This invention achieves the green synthesis of 3'-amino nucleotides through a simple and efficient "catalytic reduction-cation exchange purification" process, solving the core pain points of existing technologies. The purity and yield of the product meet the industrial application standards for pharmaceutical intermediates, and it can be widely used in the preparation of intermediates for anti-HIV drugs, cancer gene therapy drugs, and other fields, with significant economic value and social significance.

[0057] It should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for efficient green synthesis of 3'-amino nucleotides, characterized by, The target product is synthesized from 3'-azido nucleotide by the following steps: S1, dissolving 3'-azido nucleotide raw material in pure water, adding 5% Pd / C catalyst, and performing reduction reaction under hydrogen atmosphere; S2, after the reaction is completed, the catalyst is removed by filtration, the filtrate is diluted and then purified by cation exchange resin, gradient elution is performed using TEAB aqueous solution, and the eluate is concentrated and freeze-dried to obtain 3'-amino nucleotide; The synthesis route is as follows: In the formula, R1 is selected from H, OH or SH; R2 is selected from monophosphate, diphosphate, triphosphate or polyphosphate.

2. The method for efficient green synthesis of 3'-amino nucleotides according to claim 1, wherein, In step S1, the amount of 5% Pd / C catalyst used is 10wt% of the mass of 3'-azido nucleotide raw material.

3. The efficient and green synthesis method for the 3'-amino nucleotide according to claim 1, characterized in that, In step S1, the amount of pure water used is 10 times (v / w) the mass of 3'-azido nucleotide raw material, i.e. 1 mmol of raw material corresponds to 10 mL of pure water.

4. The method for efficient green synthesis of 3'-amino nucleotides according to claim 1, wherein, In step S1, the reduction reaction conditions are: reaction temperature 50°C, reaction time 2h, and hydrogen atmosphere is normal pressure hydrogen.

5. The method for efficient green synthesis of 3'-amino nucleotides according to claim 4, wherein, In step S2, diatomite is used as a filter aid for filtration, and the filtrate is diluted with pure water to a concentration of 1 mmol / L before cation exchange resin purification.

6. The efficient and green synthesis method for the 3'-amino nucleotide according to claim 1, characterized in that, In step S2, the cation exchange resin is DEAE resin; and the concentration gradient of TEAB aqueous solution is 0.1mM-0.3mM.

7. The method of synthesis according to any one of claims 1 to 6, wherein, The HPLC purity of the 3'-amino nucleotide is ≥95%, and the molar yield is ≥46%.

8. The efficient and green synthesis method for the 3'-amino nucleotide according to claim 1, characterized in that, The 3'-azido nucleotide is selected from one of 3'-N3-dATP, 3'-N3-dCMP, 3'-N3-dGDP, 3'-N3-GDP or 3'-N3-dGQP.

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