A long-chain fatty acid with precise deuterium labeling in the middle section of the carbon chain, a preparation method and application thereof

Abstract

This invention provides a method for the precise deuterium labeling of long-chain fatty acids in the mid-chain, its preparation, and its applications. The method uses commercially available and inexpensive deuterated ethylene glycol-D4 as a starting material, and involves eight core steps: protection, activation, nucleophilic substitution, deprotection, secondary activation, substitution reaction, olefin metathesis, and hydrogenation reduction. This method offers mild preparation conditions and controllable reactions, enabling precise deuterium labeling at the mid-chain sites of various long-chain and very long-chain fatty acids, with a deuteration rate of no less than 98%. Furthermore, this invention can react the obtained long-chain fatty acids with phospholipid intermediates to prepare deuterium-labeled lecithin products targeting the mid-chain, addressing the drawbacks of traditional deuterium-labeled phospholipids, such as easy metabolic shedding of the labeling site and significant interference with detection signals. It also boasts advantages such as high purity, metabolic stability, and good biocompatibility, and can be widely applied in fields such as lipidomics quantification, biochemical metabolic tracing, food adulteration identification, and pharmaceutical research, providing a precise and efficient technical tool for related studies.

Description

[0001] This application claims priority to a prior Chinese application, application number 2026100286444, filed on January 9, 2026; all its contents are part of this invention. Technical Field

[0002] This invention belongs to the field of chemical synthesis, specifically relating to a long-chain fatty acid with precise deuterium labeling in the middle segment of the carbon chain, its preparation method, and its application. Background Technology

[0003] Fatty acids are carboxylic acid compounds formed by the linkage of a carboxyl group with an aliphatic hydrocarbon group (or hydrogen atom). They are the core structural units constituting various lipid compounds such as glycerol fatty acid esters, sphingomyelin, lecithin, and cholesterol esters. In recent years, with the rapid development of mass spectrometry detection technology, liquid chromatography-tandem mass spectrometry (LC-MS / MS) has become the mainstream technique for the qualitative and quantitative analysis of lipids and other target substances. The accompanying isotope dilution mass spectrometry and stable isotope tracing techniques, with their advantages of high precision and strong anti-interference, have become core technologies with great development prospects in fields such as clinical pharmacokinetic research, molecular biology research, food safety testing, and environmental science analysis.

[0004] Deuterated fatty acids and their derivatives are not only important chemical intermediates in organic synthesis but also core functional reagents in analytical testing and life science research, with broad application prospects in medicine, materials science, analytical chemistry, and life sciences. Among them, deuterated derivatives of long-chain fatty acids, due to their excellent metabolic stability and isotope tracing properties, demonstrate extremely high application value and market potential in innovative drug development, biological metabolic pathway research, and early disease diagnosis. Given the extremely complex composition of the metabolic matrix in organisms, the deuteration labeling site of fatty acids directly determines their application effectiveness and data reliability—specific deuteration labeling at mid-chain sites of fatty acids is more conducive to achieving stable and long-term monitoring of tracers in complex biological metabolic processes, and is currently the core demand direction for the development of deuterated fatty acids.

[0005] Currently, while there are some research reports on the synthesis technology of deuterium-labeled long-chain fatty acids, the core synthetic strategies mainly focus on two categories: one is the hydrogen-deuterium (H / D) exchange reaction, and the other is the deuteration reduction reaction of unsaturated bonds. Among them, the hydrogen-deuterium exchange reaction is usually carried out under the catalysis of platinum group metal catalysts (such as Pt / C). Although it can achieve deuteration modification of fatty acids, it has core defects such as harsh reaction conditions, low deuteration abundance of products, and strong randomness of deuteration labeling sites. The deuteration reduction method of unsaturated bonds mainly uses deuteration reducing agents such as deuterium gas to reduce unsaturated fatty acid precursors to introduce deuterium atoms. For example, four deuterium atoms can be introduced at once by reducing alkynes with deuterium gas. However, alkene bond migration is very likely to occur during this reaction, which ultimately leads to the dilution of the isotopic abundance of the product or the problem of over-deuteration, making it difficult to prepare target products with specific site labeling and stable deuteration abundance.

[0006] Stefan Sonnenberger et al. reported two synthetic routes for deuterium-labeled fatty acids in "Synthesis of ceramides NS and NP with perdeuterated and specifically ω deuterated N‐acyl residues" (DOI: 10.1002 / jlcr.3443): First, using a Pd / C catalytic hydrogen-deuterium exchange reaction, n-tetracosanoic acid was treated in heavy water under high pressure conditions of 195 °C and 15 bar to prepare fully deuterated fatty acids (96% deuteration rate); Second, through a copper-catalyzed Grignard coupling reaction, the carbon chain of a ω-deuterium-labeled alkyl bromide was extended with a protecting ω-bromool, and after oxidation, a carbon chain terminal (ω-position) specifically deuterium-labeled fatty acid was obtained. However, both of the above-mentioned schemes have obvious technical limitations: the two paths can only achieve site-specific labeling at the ends of fatty acid carbon chains or random deuterium labeling of the entire carbon chain, and cannot achieve targeted labeling of specific sites in the middle of the carbon chain; at the same time, the hydrogen-deuterium exchange reaction needs to be carried out under extreme conditions of high temperature and high pressure, and is highly dependent on high-risk or special deuterium sources such as deuterium gas and heavy water, which not only makes the process difficult to operate and poses high safety risks, but also greatly limits the expansion of its application scenarios.

[0007] In summary, the industry has not yet developed a synthetic technology that can efficiently, safely, and stably achieve targeted deuteration of the carbon chain mid-chain of fatty acids. Existing methods suffer from problems such as uncontrollable labeling sites, harsh reaction conditions, low deuteration rates of products, and poor process adaptability. These problems have become the core technical bottlenecks restricting the large-scale application of deuterated long-chain fatty acids in multiple fields, and there is an urgent need to develop new synthetic methods to solve the above-mentioned industry problems. Summary of the Invention

[0008] This invention provides a method for precisely labeling long-chain fatty acids with deuterium in the mid-chain segment, its preparation method, and its applications. The preparation method uses commercially available and inexpensive deuterated ethylene glycol-D4 as a starting material and completes the preparation through eight core reaction steps: protection, activation, nucleophilic substitution, deprotection, secondary activation, substitution reaction, olefin metathesis, and hydrogenation reduction. The overall preparation conditions are mild, the reaction process is highly controllable, and precise deuterium labeling can be achieved at the mid-chain sites of long-chain and even very long-chain fatty acids of varying lengths, with a deuteration rate of not less than 98% for the obtained product. This invention can also react the long-chain fatty acids obtained above with phospholipid intermediates to prepare lecithin products with precise deuterium labeling in the middle of the carbon chain. This type of compound effectively solves the industry pain points of traditional deuterium-labeled phospholipids, such as easy loss of labeling sites and large interference in detection signals during metabolism. At the same time, it has the characteristics of high purity, excellent metabolic stability and good biocompatibility. It can be widely used in multiple fields such as lipidomics quantitative analysis, biological metabolism tracing research, food adulteration identification and pharmaceutical research and development, providing a precise and efficient technical tool for research and application in related fields.

[0009] On one hand, the present invention provides a long-chain fatty acid with precise deuterium labeling in the middle segment of the carbon chain, the structure of which is as follows: ; Where n1 and n2 are integers including 0, 1, 2, 3, ...

[0010] The deuterium-labeled long-chain fatty acids provided by this invention have deuterium labeling sites at specific sites in the middle of the fatty acid carbon chain, while the deuterium-labeled fatty acid chains prepared by existing technologies mostly have deuterium labels at the terminal (ω site) or randomly distributed deuterium labels throughout the chain, with very few targeted labels in the middle.

[0011] On the other hand, the present invention provides a method for preparing long-chain fatty acids as described above, wherein the preparation method uses deuterated diols containing at least two carbon atoms as raw materials, wherein the number of deuterated atoms is at least three, and extends the carbon chain at both ends of the raw materials.

[0012] This invention uses deuterated diols containing at least two carbon atoms as raw materials, which can serve as reaction sites for the extension of carbon chains at both ends, providing a basis for "bidirectional chain extension." This ensures that the deuterated labeling region is ultimately located in the middle of the carbon chain, meeting the requirements for targeted labeling. Having at least three deuterium atoms serves two purposes: first, it ensures the signal strength for mass spectrometry detection and metabolic tracing, avoiding interference; second, it provides redundancy to offset the loss of small amounts of deuterium in subsequent reactions, ensuring a deuteration rate of ≥98% in the final product, meeting the requirements for precise applications.

[0013] The preparation method provided by this invention extends the carbon chain at both ends of deuterated ethylene glycol-D4 according to the synthesis purpose. By adjusting the carbon chain length, i.e., the number of carbon atoms corresponding to n1 and n2 respectively, deuterium labeling can be achieved at different positions in the middle of the long aliphatic chain.

[0014] Further, extending the carbon chain at the left end of the deuterated ethylene glycol-D4 includes the following steps: (1) Deuterated ethylene glycol-D4 is subjected to a protection reaction and a halogenation reaction or a sulfonation reaction to obtain the first deuterium-containing active intermediate; (2) The first deuterium-containing active intermediate reacts with a nucleophile to obtain a deuterium-containing long-chain intermediate.

[0015] Further, in step (1), the deuterated ethylene glycol-D4 reacts with benzyl bromide under the action of silver oxide to synthesize a benzyl-protected intermediate, and then undergoes a halogenation reaction with a halogenating reagent or a sulfonating reaction with a sulfonating reagent under the action of triphenylphosphine; the halogenating reagent used includes any one of I2, NBS, NCS, and NIS, and the sulfonating reagent is TsCl; the nucleophilic reagent in step (2) includes any one of alkyl Grignard reagent and alkyl lithium reagent.

[0016] Furthermore, the reaction flow of step (1) when the carbon chain on the left end is as follows: Compound 2 was obtained from deuterated ethylene glycol-D4 (compound 1) through protection and halogenation or sulfonation reactions: ; Where X is any one of I, Br, Cl and OTs.

[0017] The halogenating or sulfonating reagents used to extend the carbon chain on the left are mild reagents that only attack the hydroxyl site without destroying the carbon chain structure. Furthermore, the selected reagents do not conflict with the deuterium atom of deuterated ethylene glycol-D4, thus avoiding the dilution of deuterium.

[0018] Furthermore, the reaction flow of step (2) when the carbon chain on the left end is as follows: Compound 2 undergoes a nucleophilic reaction to yield compound 4: ; Where n1 is an integer including 0, 1, 2, 3, ...

[0019] When extending the carbon chain at the left end, the deuterated halide intermediate in step (2) undergoes a nucleophilic reaction with an alkyl Grignard reagent or an alkyllithium reagent to achieve initial carbon chain extension. The reagent can be flexibly selected according to the target carbon chain length (alkyllithium reagent for short chains and Grignard reagent for long chains). Both reagents are strong nucleophiles and can react rapidly with the first deuterated active intermediate, with yields exceeding 80%. The nucleophilic reaction using alkyl Grignard reagents or alkyllithium reagents does not require extreme low temperatures and can be carried out at room temperature, reducing the complexity of the process.

[0020] Further, extending the carbon chain at the right end of the deuterated ethylene glycol-D4 includes the following steps: (1) The deuterium-containing long-chain intermediate is subjected to deprotection reaction and halogenation reaction or sulfonation reaction to obtain a second deuterium-containing active intermediate; (2) The second deuterium-containing active intermediate reacts with an unsaturated olefin Grignard reagent to obtain a deuterium-containing olefin intermediate; (3) The olefin containing deuterium intermediate is subjected to olefin metathesis and hydrogenation reduction reaction to obtain long-chain fatty acids with precise deuterium labeling in the middle segment of the carbon chain.

[0021] Further, the deuterium-containing long-chain intermediate in step (1) is hydrogenated under palladium carbon catalysis to generate an intermediate without the benzyl protecting group, and then halogenated or sulfonated with a sulfonating agent under the action of triphenylphosphine; the halogenating agent used includes any one of I2, NBS, NCS, and NIS, and the sulfonating agent is TsCl.

[0022] Further, the catalyst used in the olefin metathesis reaction in step (3) includes any one or more of Grubbs I, Grubbs II and Schrock; the hydrogenation reduction reaction in step (3) is carried out in the presence of a catalyst, which includes any one or more of Rh(PPh3)2Cl2, Pd / C, Pt / C and PtO2.

[0023] Furthermore, the reaction flow of step (1) when the carbon chain on the right end is extended is as follows: Compound 4 is deprotected and halogenated or sulfonated to give compound 6: Where X is any one of I, Br, Cl and OTs.

[0024] The reagents that can be used in the second halogenation reaction or sulfonation reaction in this invention are the same as those that can be used in the first halogenation reaction or sulfonation reaction. They are all mild substitution reagents with high substitution site specificity, attacking only the deprotected hydroxyl site, ensuring the precise position of the leaving group of the obtained active intermediate, and laying the groundwork for subsequent nucleophilic substitution reactions.

[0025] Furthermore, the reaction flow of step (2) when the carbon chain on the right end is extended is as follows: Compound 6 reacts with compound 7 to prepare compound 8: .

[0026] Furthermore, the reaction flow of the olefin metathesis reaction in step (3) when the carbon chain at the right end is extended is as follows: Compounds 8 and 9 undergo olefin metathesis to yield compound 10: ; Where n2 is an integer including 0, 1, 2, 3, ...

[0027] When extending the carbon chain at the right end, the olefin metathesis reaction in step (3) uses Grubbs series or Schrock catalysts to achieve directional chain extension of the olefin containing deuterium intermediates. The catalysts only catalyze the metathesis of olefin double bonds, without affecting the deuterium labeling sites and carbon chain structure. The reaction conditions are mild, and the reaction can be carried out at 40°C without the need for high temperature and high pressure.

[0028] In some methods, catalysts for olefin metathesis reactions were screened, with Grubbs II being preferred.

[0029] Furthermore, the reaction flow of the hydrogenation reduction reaction in step (3) when the carbon chain on the right end is extended is as follows: Compound 10 was reduced to give compound 11: .

[0030] In step (3) of the present invention, when extending the carbon chain at the right end, the olefin containing deuterium is converted into a saturated long-chain fatty acid through a reduction reaction. This reaction process is catalyzed by any one of the four noble metal catalysts. The catalyst only catalyzes the hydrogenation of the olefin double bond and does not attack the deuterium labeling site, thus avoiding abundance dilution. At the same time, the reaction process is mild and can be carried out at room temperature and in a 15psi hydrogen atmosphere, without the need for high temperature and high pressure.

[0031] In some methods, catalysts for the hydrogenation reduction reaction were selected, with Rh(PPh3)2Cl2 being preferred.

[0032] In existing technologies, deuterium-labeled long-chain fatty acids with the middle position of the carbon chain are generally synthesized through two methods. The first method involves reducing a pre-synthesized alkyne with deuterium gas to achieve deuterium labeling; the second method involves first constructing a deuterium label at a specific site on the carbon chain, and then attaching it to other carbon chains through coupling or substitution reactions. In the first method, the reduction of the alkyne with deuterium gas leads to dilution or over-deuteration of the product due to alkene bond migration. Therefore, obtaining a deuterated product with a specific site and high abundance using the first method is difficult, and this method also presents safety concerns. In the second method, the steric hindrance is relatively large after the middle position of the carbon chain is deuterated, making subsequent reactions to extend the carbon chain difficult, ultimately resulting in low yield and isotopic abundance. The preparation method provided by this invention has the following advantages: First, the raw materials are safe and free from steric hindrance. Commercially available and inexpensive deuterated ethylene glycol-D4 is used as the core deuterium source, eliminating the need for hazardous reagents such as deuterium gas. Furthermore, the deuterated labeling site is fixed in the early stages of synthesis through a "protection-halogenation-nucleophilic" process, and subsequent carbon chain extension (olefin metathesis, hydrogenation reduction) does not involve direct reactions at the deuterated labeling site, avoiding inefficiencies caused by steric hindrance. Second, the site is precise and the abundance is stable. By controlling the length of the carbon chains at both ends, the deuteration site is precisely located in the middle region of the carbon chain. Mild reagents are used throughout the process, without damaging the CD bonds, resulting in a low deuterium atom shedding rate and a final deuteration rate exceeding 98%. Third, the process is stable and scalable. The reaction temperature is controlled between room temperature and 40°C, with low pressure, eliminating the need for extreme equipment, low byproduct content, high yield, and small batch-to-batch yield fluctuations, meeting the needs of industrial-scale mass production. This invention adopts a "mid-chain localized deuteration-terminal retention" strategy, which solves the pain points of existing technologies such as "uncontrollable sites, harsh conditions, and poor adaptability" from a methodological perspective. It achieves precise deuteration labeling of specific sites in the middle segment under mild conditions, and the synthesis process does not involve abundance dilution.

[0033] In another aspect, the present invention provides a compound with precise deuterium labeling in the middle of the carbon chain, which is prepared by acylation reaction of the aforementioned long-chain fatty acid with precise deuterium labeling in the middle of the carbon chain with any one of fatty alcohol, phosphocholine, acylcarnitine, and polypeptide via a condensation reagent.

[0034] The compound can be applied in various fields such as medicine and food.

[0035] Furthermore, the condensation reagent includes any one of DIC, EDCI, DCC, and CDI.

[0036] Furthermore, when the acylation reaction with the precisely deuterium-labeled long-chain fatty acid in the middle segment of the carbon chain is glycerophosphate choline, the structure of the compound is as follows: ; Where n1 and n2 are integers including 0, 1, 2, 3, ...

[0037] This invention prepares phospholipid compounds with precise deuterium labeling in the carbon chain mid-chain by mid-segment targeted deuterium-labeled fatty acids and phosphocholine. The high-purity synthesis process and tunable structure of the phospholipid compounds can become key materials in the fields of isotope tracing and clinical mass spectrometry analysis, fully adapting to the needs of multiple scenarios. For example, they can be used as internal standards for precise quantitative detection in lipidomics LC-MS / MS, biological metabolism tracing research tools, internal standards for identifying adulteration of lipids in food, and auxiliary tools for pharmaceutical research and development. This solves the problems of easy detachment and large interference of traditional labeling units, and realizes the precise quantification of lipids in biological samples and food.

[0038] Furthermore, the reaction process for the precisely deuterium-labeled phospholipid compound in the middle segment of the carbon chain is as follows: Compound 11 and compound 12 react under the conditions of the condensing agent to form compound 13:

[0039] Where n1 and n2 are integers including 0, 1, 2, 3, ...

[0040] In some embodiments, when n1=13 and n2=6 of the above compound 13, the carbon chain mid-segment precisely deuterated lipid compound prepared by the method provided by the present invention is lysophosphatidylcholine, which can be used as an internal standard for newborn screening, lipidomics quantification, and identification of lipid adulteration in complex foods.

[0041] The present invention has the following advantages: 1. The deuterium labeling site in the preparation method of this invention is precisely controllable, breaking through the bottleneck of traditional technology: Through an 8-step step-by-step control process of “protection-activation-nucleophilic substitution-deprotection-secondary activation-substitution reaction-olefin metathesis-hydrogenation reduction”, by adjusting the length of the carbon chains at both ends of the deuterium source, the targeted labeling of deuterium atoms in the middle of the carbon chain can be realized and controlled, completely solving the problem of random or limited labeling sites in existing technologies (hydrogen / deuterium exchange, deuteration reduction of unsaturated bonds) that are limited to the ends of the carbon chain.

[0042] 2. The reagents and catalysts in the preparation method of this invention have excellent compatibility, ensuring product quality: mild halogenation reagents such as I2 and NBS are selected, which specifically attack hydroxyl sites without destroying the carbon chain structure and deuterium labeling sites, thus avoiding deuterium loss; Grubbs II catalyst is preferred for olefin metathesis and Rh(PPh3)2Cl2 catalyst is preferred for hydrogenation reduction, which greatly improves reaction selectivity, reduces side reactions, and results in high product yield and high isotope abundance (deuteration rate ≥98%).

[0043] 3. The raw materials used in the preparation method of this invention are safe and readily available, avoiding dependence on dangerous deuterium sources: The preparation method uses commercially available and inexpensive deuterated ethylene glycol-D4 as the core deuterium source, without the need for dangerous or demanding raw materials such as deuterium gas and D2O, making the procurement and use safer and the application scenarios unrestricted.

[0044] 4. The preparation method of this invention has mild and safe reaction conditions, which reduces the difficulty of the process: the reaction temperature is controlled at room temperature to 40°C throughout the process, and hydrogenation reduction only requires a 15psi hydrogen atmosphere, without the need for high temperature and high pressure conditions; nucleophilic reactions, halogenation reactions, etc. do not require extreme environments, the operation is simple and safe, and it is easy to scale up production.

[0045] 5. In the preparation method of this invention, the nucleophilic reaction can flexibly adjust the carbon chain length by selecting alkyl Grignard reagents (long chain) or alkyllithium reagents (short chain), and the olefin metathesis reaction further achieves directional chain elongation. The process design is compatible with the synthesis of long-chain and very long-chain fatty acids, without carbon chain length limitations, thus solving the problem of limited carbon chain extension in the prior art.

[0046] 6. The long-chain fatty acids with precise deuterium labeling in the middle of the carbon chain prepared by the present invention react with fatty alcohols, phosphocholine, acylcarnitine or polypeptides to generate compounds with precise deuterium labeling in the middle of the carbon chain. These compounds can be applied in various fields such as medicine and food.

[0047] 7. The present invention prepares a carbon chain mid-segment precisely deuterium-labeled long-chain fatty acid and glycerophosphocholine to prepare a carbon chain mid-segment precisely deuterium-labeled phospholipid compound (lysophospholipid choline). The phospholipid compound simultaneously meets the needs of multiple scenarios such as precise quantification of lipidomics, metabolic pathway tracking, identification of food adulteration, and assistance in drug development, and solves the problems of insufficient specificity and limited applicable scenarios of the labeling tools in the prior art. Attached Figure Description

[0048] Figure 1 The 1H NMR spectrum of compound 2 from Example 1; Figure 2 The 1H NMR spectrum of compound 4 in Example 1; Figure 3 The 1H NMR spectrum of compound 5 from Example 1; Figure 4 The 1H NMR spectrum of compound 6 in Example 1; Figure 5 The 1H NMR spectrum of compound 8 from Example 1; Figure 6 The 1H NMR spectrum of compound 11 (hexadecanoic acid-D4) from Example 1; Figure 7 The mass spectrum of compound 11 (hexadecanoic acid-D4) from Example 1 is shown below. Figure 8The 1H NMR spectrum of compound 13 in Example 2; Figure 9 This is the mass spectrum of compound 13 from Example 2. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0050] Example 1: Preparation of long-chain fatty acids with precise deuterium labeling in the middle segment of the carbon chain In this embodiment, deuterated ethylene glycol-D4 (compound 1) was used as a raw material to prepare long-chain fatty acids with precise deuterium labeling in the middle of the carbon chain. The preparation process is as follows: 1. Compound 2 is obtained from deuterated ethylene glycol-D4 through protection and halogenation or sulfonation reactions. Compound 2 and compound 3 then undergo a nucleophilic reaction to obtain compound 4. The reaction process is as follows: Where n1 is an integer including 0, 1, 2, 3, ...

[0051] In the above reaction process, n1=13 is taken as an example.

[0052] The synthesis steps of compound 2 are as follows: At room temperature, 12.0 g of compound 1 and 240 mL of DCM solvent were added to a 500 mL single-necked flask, followed by 34.2 g of BnBr and 62.4 g of silver oxide. The reaction was allowed to proceed for 12 hours at room temperature. After the reaction was complete, the system was filtered and concentrated to obtain a crude product. The crude product was purified by forward column chromatography (A:B = PE:EA, 330 g Flash silica gel column, approximately 10% eluent). The product fraction was collected and concentrated under reduced pressure to obtain 25.0 g of an oily substance. This oil was placed in a 1000 mL single-necked flask, and then 360 mL of DCM solvent was added. 36.3 g of PPh3 and 39.6 g of I2 (I2 can be replaced by any one of NBS, NCS, NIS, and TsCl) were slowly added. The system was then cooled to 0 °C in an ice-water bath, and then 10.9 g of imidazole was added in batches. After the addition was complete, the system was reacted at 25 °C for 12 hours. After the reaction was complete, the reaction solution was poured into an aqueous sodium thiosulfate solution and extracted with dichloromethane. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was then mixed with petroleum ether:ethyl acetate in a 1:1 ratio, filtered, and the filtrate was concentrated and purified by normal-phase column chromatography (A:B = PE:EA, 330 g Flash silica gel column). Approximately 5% of the product fraction was collected and concentrated under reduced pressure to obtain 25.0 g of an oily substance (compound 2), with a yield of 83.3% (see WO2011 / 17108, 2011, A2).1 H NMR (400MHz, Chloroform-d) δ 7.41-7.28 (m, 5H), 4.58 (s, 2H). The proton NMR spectrum of compound 2 is shown below. Figure 1 As shown.

[0053] The synthesis steps of compound 4 are as follows: At room temperature, 15.0 g of compound 2 was dissolved in 150 mL of THF. Under a nitrogen atmosphere, the mixture was cooled to 0°C in an ice-water bath. 450 mL of compound 3 was slowly added, followed by 60 mL of Li₂CuCl₄. After the addition was complete, the mixture was allowed to react at 25°C for 12 hours. After the reaction was completed, the mixture was cooled to room temperature under a nitrogen atmosphere in an ice-water bath. Ammonium chloride aqueous solution was slowly added, and ethyl acetate was added for separation. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal-phase column chromatography using a wet loading method (dissolved in DCM). The sample was loaded onto a 330 g Flash silica gel column with an A:B = PE:EA concentration. Approximately 15% of the product fraction was collected and concentrated under reduced pressure to obtain 15 g of a light yellow oil (compound 4), with a yield of 71.0%. 1 H NMR (400MHz, Chloroform- d ) δ 7.32-7.14 (m, 5H), 4.41 (s, 2H), 1.27-1.17 (m, 4H), 1.45-1.16 (m, 24H), 0.84-0.76 (m, 3H). The proton NMR spectrum of compound 4 is shown below. Figure 2 As shown.

[0054] 2. Compound 4 undergoes deprotection and halogenation or sulfonation to yield compound 6. Compound 6 reacts with compound 7 to prepare compound 8. The reaction process is as follows:

[0055] Where n1=13.

[0056] The synthesis steps of compound 5 are as follows: At room temperature, 15.0 g of compound 4 was dissolved in 225 mL of THF in a 500 mL single-necked flask, followed by the addition of 30.0 g of Pd / C. The mixture was purged three times with hydrogen gas under a hydrogen atmosphere (15 psi) at an external temperature of 40 °C for 12 hours. After the reaction was complete, the reaction solution was filtered, the filter cake was washed with THF, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal phase chromatography (A:B = PE:EA) using a 330 g Flash silica gel column, yielding approximately 10% of the product fraction. The product fraction was collected and concentrated under reduced pressure to obtain 9.0 g of a white solid (compound 5), with a yield of 98.0% and an isotopic abundance of 99.0 atom% D (compound 5 was derivatized with 2-pyridinecarboxylic acid, and the isotopic abundance was calculated using the mass cluster collection method). 1 H NMR (400 MHz, Chloroform- d ) δ 4.41 (s, 2H), 1.27-1.17 (m, 4H), 1.15-1.06 (m, 24H), 0.84-0.76 (m, 3H). The proton NMR spectrum of compound 5 is shown below. Figure 3 As shown.

[0057] The synthesis steps of compound 6 are as follows: 9.0 g of compound 5 was dissolved in 200 mL of dichloromethane, and then 5.40 g of triethylamine and 100 mg of DMAP were slowly added. Under a nitrogen atmosphere, the mixture was cooled to 0°C in an ice-water bath, and then 7.8 g of TsCl (TsCl can be replaced by any one of I2, NBS, NCS, and NIS; TsCl is preferred in this embodiment because Ts has an absorbance value under UV light, which facilitates reaction monitoring) was slowly added. After the addition was complete, the system was reacted at 25°C for 24 hours. After the reaction was completed, dichloromethane and brine were added for extraction. The aqueous phase was extracted with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal phase column chromatography (A:B = PE:EA, 330 g Flash silica gel column), with approximately 5% effluent. The product fraction was collected and concentrated under reduced pressure to obtain 12 g of white solid (compound 6), with a yield of 80.0%. 1 H NMR (400 MHz, Chloroform- d ) δ 7.85-7.74 (m, 2H), 7.34(d, J = 8.0 Hz, 2H), 2.45 (s, 3H), 1.38-1.15 (m, 24H), 0.93- 0.82 (m, 3H). The proton NMR spectrum of compound 6 is shown below. Figure 4 As shown.

[0058] The synthesis steps of compound 8 are as follows: 12 g of compound 6 was dissolved in 250 mL of tetrahydrofuran. Under a nitrogen atmosphere, the solution was cooled to 0 °C in an ice-water bath. Then, 65 mL of compound 7 and 32 mL of Li₂CuCl₄ were slowly added, and the reaction was carried out at 25 °C for 12 hours. After the reaction was completed, the reaction solution was cooled to 0 °C in an ice-water bath and slowly added dropwise to an aqueous solution of ammonium chloride. The solution was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal phase column chromatography using a dry loading method. The sample was loaded onto a 330 g Flash silica gel column with an A:B = PE:EA column and eluted with petroleum ether. The product fraction was collected and concentrated under reduced pressure to obtain 7.5 g of a colorless oil (compound 8), with a yield of 94.9%. 1H NMR (400 MHz, Chloroform-d) δ1H NMR (400 MHz, Chloroform-d) δ 5.82-5.80(m, 1H), 5.04-4.90 (m, 2H), 2.02 (d, J = 6.7 Hz, 2H), 1.34-1.16 (m, 26H),0.95-0.85 (m, 3H). The proton NMR spectrum of compound 8 is shown below. Figure 5 As shown.

[0059] 3. Compound 8 undergoes olefin metathesis to yield compound 10, and compound 10 is hydrogenated and reduced to yield compound 11. The reaction process is as follows: Where n1 = 13, and n2 is an integer including 0, 1, 2, 3, ...

[0060] In this embodiment, n2=6 is taken as an example.

[0061] The synthesis steps of compound 10 are as follows: 9.0 g of compound 8 was dissolved in 360 mL of DCM under nitrogen protection. 7.6 g of starter 9 and 3.00 g of Grubbs II catalyst (which can be replaced by either Grubbs I or Schrock catalyst) were slowly added. After addition, the reaction was carried out at 40 °C for 16 hours, and TLC was used to monitor the reaction until completion. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by normal-phase column chromatography using a dry loading method (A:B = PE:EA), 330 g Flash silica gel column, with approximately 5% elution. The product fraction was collected and concentrated under reduced pressure to obtain 6 g of a white solid (compound 10), with a yield of 40.5% and an isotopic abundance of 98.9 atom% D. The isotopic abundance was calculated using the mass cluster collection method.

[0062] The synthetic steps of compound 11 (hexadecanoic acid-D4) are as follows: At room temperature, 2.0 g of compound 10 was dissolved in 20 mL of THF in a 100 mL single-necked flask, followed by the addition of 2.0 g of Rh(PPh3)2Cl2 (which can be replaced by any of Pt / C, Pd / C, and PtO2). The mixture was purged three times with hydrogen gas, and the reaction was carried out at 25 °C for 12 hours under a hydrogen atmosphere (15 psi). After the reaction was complete, the reaction solution was filtered, the filter cake was washed with THF, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was separated by normal-phase Flash silica gel column chromatography to obtain 1.8 g of a white solid (compound 11), with a yield of 85.9% and an isotopic abundance of 98.9 atom% D. The isotopic abundance was calculated using the mass cluster collection method. [MH] - = 399.4, 1 HNMR (400 MHz, Chloroform- d ) δ 2.35 (t, J = 7.5 Hz, 2H), 1.69-1.57 (m, 2H), 1.25 (s, 39H), 0.88 (t, J = 6.7 Hz, 3H). The proton NMR spectrum of compound 11 is shown below. Figure 6 As shown.

[0063] The mass spectrum of compound 11 is as follows: Figure 7 As shown.

[0064] Example 2: Preparation of deuterium-labeled lipid compounds The hexacosanoic acid-D4 (compound 11) prepared in Example 1 can be used to prepare lipid compounds with precise deuterium labeling in the middle of the carbon chain. The reaction procedure is as follows: Where n1=13 and n2=6.

[0065] The synthesis steps of compound 13 are as follows: At room temperature, 1.0 g of compound 11 was dissolved in 100 mL of THF and 100 mL of chloroform in a 500 mL single-necked flask. Then, 1.5 g of compound 12 (glycerophosphocholine), 725 mg of DIC, and 915 mg of DMAP were added, and the reaction was carried out at 70 °C for 48 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal phase chromatography (A:B = DCM:MeOH), using a 20 g Flash silica gel column. Approximately 80% of the product fraction was collected and concentrated under reduced pressure to obtain 200 mg of a white solid (compound 13, lysophosphatidylcholine), with a yield of 10.1% and an isotopic abundance of 98.9 atom% D. The isotopic abundance was calculated using the mass cluster collection method. [M+H]+ = 640.5, 1 H NMR (400 MHz, Methanol- d 4) δ 4.29 (ddd, J =7.2, 4.8, 2.4 Hz, 2H), 4.17 (d, J = 4.4 Hz, 1H), 4.12 (d, J = 6.4 Hz, 1H), 3.97 (m, 1H), 3.91 (dt, J = 6.8, 2.4 Hz, 2H), 3.66-3.61 (m, 2H), 3.32 (s,9H),2.36 (t, J = 7.6 Hz, 2H), 1.70-1.59 (m, 2H), 1.42-1.26 (m, 40H), 0.91 (t, J =6.8 Hz, 3H). The proton NMR spectrum of compound 13 is shown below. Figure 8 As shown.

[0066] The mass spectrum of compound 13 is as follows: Figure 9 As shown.

[0067] Example 3: Screening of catalysts in the synthesis of compound 10 In Example 1, the catalyst used in the synthesis of compound 10 was Grubbs II. In this example, the type of catalyst was changed, and the other catalysts were Grubbs I and Schrock. The yield and isotopic abundance of compound 10 were compared when different catalysts were used to synthesize compound 10.

[0068] Compound 10 was synthesized using Grubbs II, Grubbs I, and Schrock as catalysts according to the steps in Example 1. The yields and isotopic abundances of compound 10 prepared with different catalysts are shown in Table 1 below.

[0069] Table 1. Yields and isotopic abundances of compound 10 prepared with different catalysts

[0070] As shown in Table 1, when Grubbs II, Grubbs I, and Schrock were used as catalysts, the final compound 10 obtained all exhibited good yields and isotopic abundances. However, the yield and isotopic abundance of compound 10 prepared using Grubbs II as a catalyst were significantly higher. This is because, compared to other catalysts, Grubbs II has more moderate reactivity, which is beneficial for improving reaction selectivity and reducing side reactions, thereby significantly increasing the product yield.

[0071] Therefore, the catalyst used in the preparation of compound 10 is preferably Grubbs II.

[0072] Example 4: Screening of catalysts in the synthesis of compound 11 In Example 1, the catalyst used in the synthesis of compound 11 (tetrahexadecanoic acid-D4) was Rh(PPh3)2Cl2. In this example, the type of catalyst was changed, and the other catalysts were Pd / C, Pt / C and PtO2, respectively. The yield and isotopic abundance of compound 11 were compared when different catalysts were used to synthesize compound 11.

[0073] Compound 11 was synthesized according to the steps in Example 1 using Rh(PPh3)2Cl2, Pd / C, Pt / C and PtO2 as catalysts, respectively. The yields and isotopic abundances of compound 11 prepared by different catalysts are shown in Table 2 below.

[0074] Table 2. Yields and isotopic abundances of compound 11 prepared with different catalysts

[0075] As shown in Table 2, when Rh(PPh3)2Cl2, Pd / C, Pt / C, and PtO2 were used as catalysts, the final compound 11 obtained all exhibited good yields and isotopic abundances. However, compared to other catalysts, the compound 11 prepared using Rh(PPh3)2Cl2 as a catalyst had a higher yield and isotopic abundance. This is because Rh(PPh3)2Cl2 has a more moderate reactivity than other catalysts, which is beneficial for improving the selectivity of the reaction and reducing the occurrence of side reactions, thereby significantly improving the yield and isotopic abundance of the product.

[0076] Therefore, the catalyst used in the preparation of compound 11 is preferably Rh(PPh3)2Cl2.

[0077] Example 5: Comparison of the effects of different preparation methods on the preparation of long-chain fatty acids with precise deuterium labeling in the middle segment of the carbon chain. In Example 1, hexacosanoic acid-D4 with targeted labeling at the middle position of the carbon chain was synthesized using the preparation method provided by the present invention.

[0078] In this embodiment, different methods were used to synthesize the hexadecanoic acid-D4, and the yields and isotopic abundances of different preparation methods were compared.

[0079] Method 1: Prepared according to the method in Example 1.

[0080] Method 2: Following the method described by John H. Horner et al. in "(v-2,v-2,v-3,v-3)-Tetradeuterio-fatty acids for mechanistic studies of enzyme-catalyzed hydroxylation reactions" (Labelled Compounds and Radiopharmaceuticals, 2012, DOI: 10.1002 / jlcr.2959.), this method primarily involves deuterium labeling via the reduction of a pre-synthesized alkyne with deuterium gas. The synthetic route is as follows: Where n1=13 and n2=6.

[0081] Method 3: First, construct a D-label at a certain site on the carbon chain, and then link it to other carbon chains through coupling or substitution reactions. The synthetic route is as follows: .

[0082] Table 3 below shows the yield and isotopic abundance of the target product prepared by the three different methods described above.

[0083] Table 3. Yields and isotopic abundances of precisely deuterium-labeled hexacosanoic acid-D4 prepared by different methods in the mid-carbon chain

[0084] As shown in Table 3, the yield and isotopic abundance of Method 1 are significantly higher than those of Methods 2 and 3. Although the yield of Method 2 is similar to that of Method 1, it has the following drawbacks: First, the alkene bonds undergo random migration during the reduction process, resulting in the inability of the deuterium atom to be precisely fixed in the middle of the carbon chain, ultimately leading to a decrease in isotopic abundance; second, the reaction conditions are harsh and dangerous, requiring 120℃ and 20 bar high pressure; third, there are many byproducts that are difficult to separate, as some alkyne bonds are over-reduced or incompletely reduced during the reduction process, generating many byproducts, requiring multiple column chromatography analyses for subsequent purification, further reducing the yield. Method 3 has the following drawbacks: First, steric hindrance leads to low coupling efficiency. The steric hindrance of the deuterium-labeled site (CD bond) is greater than that of ordinary CH bonds, making it difficult for the catalyst to approach the reaction site during the coupling reaction. This results in low conversion rate of the carbon chain extension step, ultimately leading to low yield of the target product and inability to monitor the deuteration rate. Second, deuteration abundance is easily diluted. During the coupling process, the deuterium-labeled site may be replaced by protons in the reaction system (such as H2O in the solvent or active hydrogen in the reaction reagent), resulting in a low deuteration rate. Third, batch stability is poor. The influence of steric hindrance is random, and the coupling efficiency fluctuates greatly between different batches of reactions. It is difficult to control the consistency of the carbon chain length of the product and the accuracy of the deuteration site, which cannot meet the requirements for reagent uniformity in scenarios such as detection of internal standards and metabolic tracing. Method 1 addresses the core shortcomings of Methods 2 and 3: First, the raw materials are safe and free from steric hindrance. Using deuterated ethylene glycol-D4 as the core deuterium source, it eliminates the need for hazardous reagents such as deuterium gas. Furthermore, the deuterated labeling site is fixed in the early stages of synthesis through a "protection-halogenation-nucleophilic" process, and subsequent carbon chain extension (olefin metathesis, hydrogenation reduction) does not involve direct reactions at the deuterated labeling site, thus avoiding inefficiencies caused by steric hindrance. Second, the site is precise and the abundance is stable. By controlling the length of the carbon chains at both ends, the deuteration site is precisely located in the middle region of the carbon chain. Mild reagents are used throughout the process, without damaging the CD bond, resulting in a low deuterium atom shedding rate and a final deuteration rate of over 98 atom% D. Third, the process is mild and scalable. The reaction temperature is controlled between room temperature and 40°C, with low pressure, eliminating the need for extreme equipment, low by-product content, high yield, and small batch-to-batch yield fluctuations, meeting the needs of industrial-scale mass production.

[0085] Example 6: Screening of activating reagents during the synthesis of compound 2 In Example 1, the halogenation / sulfonation step of the synthesis of compound 2 used I2 as the activating agent. In this example, the type of activating agent is changed, and other activating agents are NBS, NCS, NIS and TsCl. The yield, isotopic abundance and reaction compatibility of different activating agents used to synthesize compound 2 are compared to provide a basis for process optimization and large-scale production.

[0086] I2, NBS, NCS, NIS, and TsCl were used as activating reagents, and parallel experiments were carried out according to the synthesis steps of compound 2 in Example 1. The I2, NBS, NCS, and NIS groups were all equipped with a triphenylphosphine (PPh3) and imidazole reaction system, while the TsCl group was equipped with a triethylamine and DMAP reaction system. Except for the types of activating reagents and supporting agents, the molar ratio of raw materials, reaction temperature, reaction time, post-treatment, and purification methods were kept completely consistent.

[0087] The yields and isotopic abundances of compound 2 prepared with different activating reagents are shown in Table 4 below.

[0088] Table 4. Yields and isotopic abundances of compound 2 prepared with different activating reagents

[0089] As shown in Table 4, the choice of halogenation / sulfonation activating reagent during the synthesis of compound 2 mainly affects the yield of the target product and the compatibility with subsequent processes, while having a minimal impact on the isotopic abundance of the product. The isotopic abundance of all experimental groups remained stable above 98.8 atom% D, confirming that the activation reaction system selected in this invention does not destroy the CD bond of the deuteration site, and there is no significant loss of deuterium atoms, which can ensure the stability of the product isotopic abundance throughout the process.

[0090] I2 and NIS are the best in terms of overall performance. When I2 is used as the activator, the target product yield is the highest and the iodinated intermediate has the best leaving activity, which can ensure the high conversion rate of subsequent nucleophilic substitution reactions. It has fewer side reactions and strong process stability, making it the first choice for large-scale synthesis. The iodinated products prepared by NIS have good selectivity and fast reaction rate. Its overall performance is close to that of I2, and it can be used as an alternative reagent for iodination reactions to meet different raw material procurement and process adjustment needs.

[0091] NBS and TsCl offer distinct advantages in adaptability to different scenarios, making them preferred alternatives. Brominated products prepared by NBS exhibit good chemical stability, mild reaction conditions, few byproducts, and low subsequent purification difficulty, balancing reactivity and product storage stability, making them suitable for synthetic scenarios requiring stable intermediate storage periods and batch-to-batch stability. P-Toluenesulfonate intermediates prepared by TsCl exhibit characteristic absorption under UV light, facilitating convenient monitoring of the reaction process. Furthermore, their leaving group exhibits excellent activity, directly adaptable to subsequent nucleophilic substitution reactions, making them particularly suitable for process parameter optimization in small-scale laboratory research.

[0092] The overall performance of NCS is insufficient and it does not have the value for large-scale application. The yield of the product prepared by it is significantly lower than that of other experimental groups. The core reason is that the low chlorination reactivity leads to incomplete conversion of raw materials and high impurity content in crude product. At the same time, the weak activity of the leaving group of the generated chlorinated product will directly reduce the reaction efficiency of the subsequent nucleophilic substitution step, which cannot meet the process requirements for carbon chain extension in this invention.

[0093] In summary, I2 is the preferred activating agent for this step, as it achieves the highest product yield and optimal compatibility with the entire reaction process. However, NIS, NBS, and TsCl can be flexibly selected as alternative reagents based on the actual needs of research and development and production.

[0094] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A long-chain fatty acid with precise deuterium labeling in the middle segment of its carbon chain, characterized in that, The structure of the long-chain fatty acid is as follows: ； Where n1 and n2 are integers including 0, 1, 2, 3, ...

2. The method for preparing long-chain fatty acids as described in claim 1, characterized in that, The preparation method uses deuterated diols containing at least two carbon atoms as raw materials, wherein the number of deuterated atoms is at least three, and the carbon chains are extended at both ends of the raw materials.

3. The method for preparing long-chain fatty acids as described in claim 2, characterized in that, Extending the carbon chain at the left end of the deuterated ethylene glycol-D4 includes the following steps: (1) Deuterated ethylene glycol-D4 is subjected to a protection reaction and a halogenation reaction or a sulfonation reaction to obtain the first deuterium-containing active intermediate; (2) The first deuterium-containing active intermediate reacts with a nucleophile to obtain a deuterium-containing long-chain intermediate.

4. The method for preparing long-chain fatty acids as described in claim 3, characterized in that, Extending the carbon chain at the right end of the deuterated ethylene glycol-D4 includes the following steps: (1) The deuterium-containing long-chain intermediate is subjected to deprotection reaction and halogenation reaction or sulfonation reaction to obtain a second deuterium-containing active intermediate; (2) The second deuterium-containing active intermediate reacts with an unsaturated olefin Grignard reagent to obtain a deuterium-containing olefin intermediate; (3) The olefin containing deuterium intermediate is subjected to olefin metathesis and hydrogenation reduction reaction to obtain long-chain fatty acids with precise deuterium labeling in the middle segment of the carbon chain.

5. The preparation method according to claim 3, characterized in that, In step (1), the deuterated ethylene glycol-D4 reacts with benzyl bromide under the action of silver oxide to synthesize a benzyl-protected intermediate, and then undergoes a halogenation reaction with a halogenating reagent or a sulfonating reaction with a sulfonating reagent under the action of triphenylphosphine; the halogenating reagent used includes any one of I2, NBS, NCS, and NIS, and the sulfonating reagent is TsCl; the nucleophilic reagent in step (2) includes any one of alkyl Grignard reagent and alkyl lithium reagent.

6. The preparation method according to claim 4, characterized in that, In step (1), the deuterium-containing long-chain intermediate is hydrogenated under palladium carbon catalysis to generate an intermediate that has lost the benzyl protecting group. Then, it undergoes a halogenation reaction with a halogenating reagent or a sulfonating reaction with a sulfonating reagent under the action of triphenylphosphine. The halogenating reagent used includes any one of I2, NBS, NCS, and NIS, and the sulfonating reagent is TsCl.

7. The preparation method according to claim 4, characterized in that, The catalyst used in the olefin metathesis reaction in step (3) includes any one or more of Grubbs I, Grubbs II and Schrock; the hydrogenation reduction reaction in step (3) is carried out in the presence of a catalyst, which includes any one or more of Rh(PPh3)2Cl2, Pd / C, Pt / C and PtO2.

8. A compound with precise deuterium labeling in the mid-segment of a carbon chain, characterized in that, Prepared by acylation reaction of long-chain fatty acids with precise deuterium labeling in the middle segment of the carbon chain as described in claim 1 with any one of fatty alcohols, phosphocholine, acylcarnitine, and polypeptides via a condensation reagent.

9. The compound with precise deuterium labeling in the middle segment of the carbon chain as described in claim 8, characterized in that, The condensation reagent includes any one of DIC, EDCI, DCC, and CDI.

10. The compound with precise deuterium labeling in the middle segment of the carbon chain as described in claim 8, characterized in that, When the acylation reaction with glycerophosphate choline occurs with the precisely deuterium-labeled long-chain fatty acid in the middle segment of the carbon chain, the structure of the compound is as follows: ； Where n1 and n2 are integers including 0, 1, 2, 3, ...

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