A method for catalyzing acetal reactions using bovine trypsinogen

Abstract

This invention discloses a method for catalyzing acetal reactions using bovine trypsinogen, belonging to the field of organic chemical engineering. Experiments revealed that bovine trypsinogen possesses two catalytic activities: one is a natural-like activity catalyzing transesterification reactions, and the other is a hybrid activity catalyzing acetal reactions. The results demonstrate that bovine trypsinogen has better catalytic ability than bovine trypsin, amino acids, or other proteins, and can be used to catalyze acetal reactions, including tandem reactions of transesterification and acetalization, thus solving the problems of low reaction efficiency, harsh reaction conditions, difficult operation, and severe pollution associated with existing acetal reactions.

Description

Technical Field

[0001] This invention belongs to the field of organic chemical technology, specifically relating to a method for catalyzing acetal reactions using bovine trypsinogen. Background Technology

[0002] Biocatalysis is a catalytic technology that uses enzymes or enzyme-containing biological organisms (such as organelles, whole cells, and tissues) as catalysts to drive reactions. With its core advantages of being environmentally degradable, having mild reaction conditions, high catalytic efficiency, and being environmentally friendly, it has become an important supplementary means in the field of chemical synthesis and one of the best alternative technologies to traditional chemical catalysis. It is in line with the current concept of green and sustainable development, and its application potential in fields such as chemical engineering and medicine continues to stand out.

[0003] Enzymes, as the core catalytic units of biocatalysis, possess outstanding advantages such as degradability, mild reaction conditions, and recyclability, making them a key supplement and optimal alternative to traditional chemical catalysts. In recent years, research has revealed that an increasing number of enzymes exhibit "hybridity"—that is, they can accept a variety of non-natural substrates and catalyze different types of reactions. This characteristic significantly expands the application boundaries of enzymes. Among them, trypsin, an important member of the serine hydrolase family, has its natural catalytic activity in hydrolyzing peptide bonds formed at the carboxyl terminus of lysine / arginine in proteins. Recent studies have further confirmed that trypsin also possesses hybridity, catalyzing aldol reactions and C(sp...) reactions. 3 Trypsin undergoes various organic synthesis reactions, including 1H-functionalization, Biginelli reaction, and Mannich reaction. From a structural transformation perspective, trypsin is secreted from the pancreas as inactive trypsinogen into the duodenum. After specific cleavage of the highly conserved N-terminal activating peptide by enterokinase, it undergoes a conformational change: the newly formed N-terminal Ile16 interacts with Asp194, inducing a well-defined substrate-binding site and an oxyanion hole, resulting in a more compact enzyme structure and significantly increased rigidity, ultimately transforming into highly active mature trypsin. However, trypsinogen itself is generally considered to have no natural catalytic activity, and there are currently no reports of using trypsinogen to catalyze organic synthesis reactions.

[0004] In modern organic synthetic chemistry and fine chemical engineering, the acetal reaction is the process by which aldehydes react with alcohols to form acetals. This reaction can temporarily mask the aldehyde group, and its reversibility meets the needs of multi-step synthesis, making it widely used. The O,O-acetal reaction is one of the main methods in protecting group chemistry for effectively masking highly reactive aldehyde functional groups. In multi-step chemical transformations, through protection and deprotection operations, the aldehyde group can be made to participate in the reaction only in certain specific stages. Furthermore, many pharmaceutical and natural product molecules contain O,O-acetal structural units in their skeletons, and these compounds are also extremely important industrial chemicals such as diesel fuel additives, food and cosmetic flavorings, and organic reaction intermediates. Due to the various practical uses of acetal compounds, corresponding synthetic methods have been reported, mainly involving improvements in catalysts, including strong inorganic acids, Lewis acids containing transition metals, strong organic acids, and strong bases. However, these chemical catalytic processes still have many problems, such as the use of flammable, toxic, or highly corrosive reagents; the need for high temperatures and inert gas atmospheres or special reaction equipment. These factors not only increase the difficulty of practical operation but also pollute the environment. Therefore, it is necessary to develop greener, more efficient, and milder synthesis methods. 1,1-Dimethoxyethane (also known as acetaldehyde dimethyl acetal) is an important oxygen-containing reagent used in the large-scale production of various fine chemicals such as pharmaceuticals, artificial fragrances, and organic synthesis intermediates. For example, it can be used to prepare vinyl methyl ether via gas-phase cracking. In addition, it can also be used directly as a diesel additive to improve combustion efficiency or as a battery electrolyte to improve the electrochemical performance of electrode materials. Given the multiple practical uses of this compound, the development of corresponding preparation methods has received widespread attention from academia and industry. Common methods include the direct acetalization reaction of acetaldehyde and methanol using liquid acids (sulfuric acid, hydrochloric acid, and p-toluenesulfonic acid, etc.), solid acids (acidic zeolites, molecular sieves, and ion exchange resins, etc.), and ionic liquids as catalysts. However, acetaldehyde has a low boiling point, an irritating odor, and is prone to side reactions such as polymerization and oxidation; secondly, acidic catalysts generally suffer from equipment corrosion problems, which are not conducive to long-term stable operation. Other methods, such as transition metal catalysis of acetylene or reaction of syngas with methanol to prepare 1,1-dimethoxyethane, not only pose a greater risk of environmental pollution, but also significantly increase the operational difficulty by using gaseous feedstocks.

[0005] In summary, while acetal reactions (or reactions involving acetal reactions) play an indispensable role in organic synthesis, existing preparation methods generally suffer from problems such as harsh reaction conditions, highly hazardous reagents, demanding equipment requirements, and poor environmental compatibility. Therefore, developing greener, simpler, more efficient, and milder synthetic techniques is of great significance for promoting the development of organic synthetic chemistry and upgrading the fine chemical industry, and is also a key issue that urgently needs to be addressed by both academia and industry. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide a method for acetal reaction catalyzed by bovine trypsinogen, so as to solve the technical problems of low reaction efficiency, harsh reaction conditions, difficult operation and serious pollution in the existing acetal reaction.

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

[0008] The first aspect of the present invention discloses a method for catalyzing an acetal reaction using bovine trypsinogen as a catalyst.

[0009] Preferably, bovine trypsinogen is used to catalyze the acetal reaction between aromatic aldehydes and a methanol-containing solution to obtain O,O-acetal.

[0010] Preferably, the aromatic aldehyde is benzaldehyde or its derivatives.

[0011] Preferably, the ratio of aromatic aldehyde to methanol is 0.05 mmol: (210~300) μL.

[0012] Preferably, the ratio of aromatic aldehyde, methanol and bovine trypsinogen is 0.05 mmol: (210~300) μL: (0.5~5.0) mg.

[0013] Preferably, the reaction temperature is 37℃~60℃ and the reaction time is 1~12 h.

[0014] Preferably, in the methanol-containing solution, the volume fraction of methanol is >70%.

[0015] Preferably, the reaction temperature is 50℃~60℃ and the reaction time is 6~12 h.

[0016] Preferably, p-nitrobenzaldehyde, pure methanol, and bovine trypsinogen are mixed and reacted at 50°C for 6 h to obtain O,O-acetal.

[0017] Preferably, bovine trypsinogen is used as a catalyst to catalyze the tandem reaction of transesterification and acetalization.

[0018] More preferably, bovine trypsinogen catalyzes the transesterification reaction of ethylene ester with a methanol-containing solution to obtain vinyl alcohol, which is spontaneously converted to acetaldehyde in situ; acetaldehyde undergoes an acetal reaction with a methanol-containing solution under the further catalysis of bovine trypsinogen to obtain 1,1-dimethoxyethane.

[0019] Preferably, the volume ratio of ethylene ester to methanol is 1:(5~29).

[0020] Preferably, the ratio of ethylene ester, methanol and bovine trypsinogen is 1 μL:(5~29) μL:(0.5~4.0) mg.

[0021] Preferably, the vinyl ester is vinyl acetate, vinyl propionate, vinyl butyrate, or vinyl hexanoate.

[0022] Preferably, the methanol-containing solution is a pure methanol solution, a methanol-ethanol mixture, a methanol-n-propanol mixture, a methanol-isopropanol mixture, a methanol-n-butanol mixture, or a methanol-isobutanol mixture.

[0023] Preferably, the methanol-containing solution has a volume fraction of ≥54.5%.

[0024] Preferably, the volume of the methanol-containing solution is greater than the volume of the ethylene ester.

[0025] Preferably, the reaction temperature is 37℃~60℃ and the reaction time is 24~144 h.

[0026] Preferably, vinyl acetate, methanol and bovine trypsinogen are mixed and reacted at 50°C for 72 h to obtain 1,1-dimethoxyethane.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention provides a method for catalyzing acetal reactions using bovine trypsinogen. Experiments have shown that bovine trypsinogen exhibits mixed activity in catalyzing acetal reactions. Therefore, using bovine trypsinogen as a catalyst in organic synthesis reactions involving acetal reactions provides mild reaction conditions, eliminates the need for flammable, toxic, or highly corrosive reagents, and is environmentally friendly.

[0029] Furthermore, the synthesis of O,O-acetal based on the catalytic activity of bovine trypsinogen offers a more environmentally friendly and simpler approach compared to traditional chemical methods. It avoids the use of flammable, toxic, or corrosive reagents such as strong inorganic acids, Lewis acids containing transition metals, strong organic acids, and strong bases. The reaction conditions are also milder, requiring no high temperatures, inert gas atmospheres, or special reaction apparatus, significantly reducing the difficulty of practical operation and effectively minimizing the risk of environmental pollution. In addition, this method can also achieve good yields in the synthesis of aldehydes with unique structures, such as those with aliphatic chains, acid-sensitive unsaturated bonds, and protecting groups. In particular, when using bovine trypsinogen as a catalyst and benzaldehyde and its derivatives, along with methanol, as reactants to prepare O,O-acetal, the yield can reach up to 98%, demonstrating highly efficient catalytic activity.

[0030] Furthermore, based on the catalytic activity of bovine trypsinogen, a tandem reaction of transesterification and acetalization was carried out. Experiments revealed that bovine trypsinogen possesses two catalytic activities: one is a natural-like activity catalyzing transesterification, and the other is a hybrid activity catalyzing acetalization. Therefore, bovine trypsinogen can serve as a catalyst for the tandem reaction of transesterification and acetalization, solving the technical problems of harsh reaction conditions, difficult operation, and severe pollution associated with traditional transesterification and acetalization reactions.

[0031] Furthermore, based on the catalytic activity of bovine trypsinogen, 1,1-dimethoxyethane was prepared via a one-pot tandem reaction. First, bovine trypsinogen, exhibiting a trypsin-like natural activity, catalyzes the transesterification reaction between vinyl acetate and methanol. The resulting vinyl alcohol is unstable and spontaneously prepares acetaldehyde in situ. Then, acetaldehyde further reacts with methanol in an acetal reaction under the action of bovine trypsinogen to obtain the target product. In this process, bovine trypsinogen simultaneously exhibits two catalytic activities: one is a natural-like activity catalyzing the transesterification reaction, and the other is a hybrid activity catalyzing the acetal reaction. Methanol also plays multiple roles, serving as both a raw material and a medium for the two-step reaction. Therefore, this method avoids the use of chemical catalysts with high environmental pollution risks. The reaction conditions are milder than traditional chemical methods, requiring no high temperature or pressure, and avoids the direct use of low-boiling-point, highly toxic acetaldehyde or gaseous reactants (such as acetylene and syngas), achieving a yield of up to 63%. Attached Figure Description

[0032] Figure 1 The flowchart of the acetal reaction catalyzed by bovine trypsinogen is shown in the present invention; wherein, (A) is the acetal reaction; and (B) is the tandem reaction of transesterification and acetal reaction.

[0033] Figure 2 The 1H NMR spectrum of the O,O-acetal prepared in this invention is shown.

[0034] Figure 3 The carbon NMR spectrum of the O,O-acetal prepared in this invention;

[0035] Figure 4 This is a graph showing the change in reaction yield over time at different temperatures in the acetal reaction catalyzed by bovine trypsinogen according to the present invention.

[0036] Figure 5 The nuclear magnetic resonance hydrogen spectrum of the reaction mixture of 1,1-dimethoxyethane prepared in this invention;

[0037] Figure 6 The reaction mixture of 1,1-dimethoxyethane prepared in this invention is shown in the gas chromatogram obtained by gas chromatography-mass spectrometry.

[0038] Figure 7 The mass spectrum of the reaction mixture of 1,1-dimethoxyethane prepared in this invention was obtained by gas chromatography-mass spectrometry. Detailed Implementation

[0039] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0040] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0041] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0042] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0043] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0044] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0045] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Bovine serum albumin, bovine trypsin, and bovine trypsinogen were used in the following examples and were all purchased from Sigma-Aldrich.® The company, with product numbers A1933, T1426 and T1143, uses other chemical reagents that, unless otherwise stated, are conventional commercially available products with specifications that are standard in the field.

[0046] I. Acetal reaction catalyzed by bovine trypsinogen.

[0047] like Figure 1 As shown in (A), an aromatic aldehyde, a methanol-containing solution, and bovine trypsinogen are mixed to obtain O,O-acetal;

[0048] In the methanol-containing solution, the volume fraction of methanol is >70%, preferably pure methanol; the reaction temperature is 37℃~60℃, and the reaction time is 1~12 h.

[0049] 1. Preparation of O,O-acetal using bovine trypsinogen as a catalyst.

[0050] Example 1

[0051]

[0052] 0.05 mmol of p-nitrobenzaldehyde, 300 μL of pure methanol and 3.0 mg of bovine trypsinogen were mixed and reacted in a shaker at 50 °C and 200 rpm for 6 h to obtain O,O-acetal.

[0053] Compounds were characterized by proton and carbon NMR spectra. Proton NMR (1H NMR) spectrum (… 1 H NMR and carbon spectroscopy (H NMR) 13 CNMR detection conditions: Agilent DD2-500 MHz nuclear magnetic resonance spectrometer, DMSO- d 6 is the solvent. The results of the proton and carbon NMR spectra are as follows: Figure 2 and Figure 3 As shown.

[0054] NMR characterization data of O,O-acetal: 1 H NMR (500 MHz, DMSO- d 6): δ 8.21 (d, J = 8.2Hz, 2H), 7.63 (d, J = 8.1 Hz, 2H), 5.50 (s, 1H), 3.25 (d, J = 1.2 Hz, 6H). 13 CNMR (125 MHz, DMSO- d6) δ 148.0, 145.8, 128.4, 123.8, 101.9, 53.3, 40.5,40.3, 40.1, 40.0, 39.8, 39.6, 39.5.

[0055] The yield of the compound was determined by high performance liquid chromatography (HPLC). The analytical conditions were: Agilent 1260 Infinity HPLC system, Eclipse Plus C18 reversed-phase column (4.6 × 150 mm, 3.5 μm), acetonitrile:water = 80:20, column temperature 25°C, flow rate 1.0 mL / min, detection wavelength λ = 254 nm, injection volume 1.0 μL, and yield 76%.

[0056] 2. Investigation of reaction conditions.

[0057] 1) The effect of different catalysts on the acetal reaction.

[0058] To verify the ability of bovine trypsinogen to catalyze the O,O-acetal reaction, p-nitrobenzaldehyde and methanol were used as templates and reactants, with methanol also serving as the reaction medium. Different catalysts were used to investigate the effects of different catalysts on the acetal reaction.

[0059] Table 1. Effect of different catalysts on acetal reaction

[0060]

[0061] As shown in Table 1, referring to Example 1, bovine trypsinogen was replaced with bovine trypsin, bovine serum albumin, no catalyst was added, and a catalytic triplet amino acid combination (aspartic acid, histidine, and serine), imidazole, serine, aspartic acid, and histidine were prepared to prepare Comparative Examples 1 to 8.

[0062] Compared with Comparative Examples 1 to 8, it can be seen that under the same reaction conditions, the yield using bovine trypsinogen was as high as 76%, while the yield of highly active natural mature bovine trypsinogen was only 17%, far lower than that of bovine trypsinogen. The reaction performance was extremely poor with bovine serum albumin, a catalytic triplet of amino acids (aspartic acid, histidine, and serine), imidazole as catalysts, or in the absence of a catalyst. These results rule out the possibility of non-specific catalytic reactions of amino acid side chains.

[0063] 2) The effect of water content on acetal reaction.

[0064] In organic media, water molecules act as a "lubricant" for enzyme conformation, greatly affecting the activity of enzyme-catalyzed reactions. The effect of water content on acetal reactions will be further investigated below.

[0065] Table 2 Effect of water content on acetal reaction

[0066]

[0067] The results are shown in Table 2. It can be observed that the yield decreases sharply with increasing water content. When the water content is 1% of the total volume, the yield drops from 76% to 63% (Example 2); and when the water content increases to 50%, the yield is only 5% (Example 7). This is because the formation and hydrolysis of O,O-acetal is a dynamic equilibrium reaction, and increasing the water content significantly inhibits the forward reaction. Therefore, using anhydrous methanol as the solvent and a single solvent environment also helps to improve the reaction yield.

[0068] 3) Effect of bovine trypsinogen dosage on acetal reaction.

[0069] Table 3 Effect of bovine trypsinogen dosage on acetal reaction

[0070]

[0071] As shown in Table 3, referring to Example 1, the amount of bovine trypsinogen was adjusted to prepare Examples 8 to 10. It can be seen that as the enzyme amount increased from 0.5 mg to 5.0 mg, the yield gradually increased, reaching a maximum of 83%. However, the yield increase was greatest (26%) when the enzyme amount was 1.0 mg compared to 0.5 mg; further increasing the enzyme amount only increased the yield by about 10%. Therefore, 1.0 mg of bovine trypsinogen was selected for further optimization.

[0072] 4) The effect of reaction temperature on acetal reaction.

[0073] Temperature is a crucial factor affecting the efficiency of enzyme-catalyzed reactions. Next, we will investigate the trend of template reaction yield over time under three temperature conditions.

[0074] Table 4. Effects of reaction temperature and time on acetal reaction

[0075]

[0076] The results are shown in Table 4 and Figure 4 As shown, increasing the temperature is beneficial to the reaction. At 37°C and 50°C, the yield gradually increases with the extension of reaction time, reaching its maximum at 12 hours. At 60°C, an 88% yield can be obtained after 8 hours of reaction (Example 25), and further increasing the reaction time does not change the yield much.

[0077] 5) The effect of reaction substrate on acetal reaction.

[0078] Based on the above optimization experiments, the optimal experimental conditions for the mixed-catalyzed O,O-acetal reaction of bovine trypsinogen were finally determined: 0.05 mmol of p-nitrobenzaldehyde, 300 μL of methanol, and 1.0 mg of bovine trypsinogen, reacted at 60℃ for 8 h, with a yield of 88% (3a). Furthermore, the universality of the substrate for acetal reactions was investigated.

[0079]

[0080] Replacing p-nitrobenzaldehyde with ortho- and meta-substituted nitrobenzaldehyde yielded 96% and 75% yields, respectively (3b, 3c). Then, replacing methanol with ethanol to react with the ortho-, meta-substituted nitrobenzaldehyde also yielded good yields of 65%, 72%, and 70% (3d, 3e, 3f), but the yields were lower compared to those with methanol.

[0081]

[0082] Next, we reacted different aromatic aldehydes with methanol and found that para-substituted benzaldehydes, such as those with cyano, chlorine, and bromine groups, achieved yields close to 100% (3g, 3h, 3l). Benzaldehydes with monohalogen or dichloro substitution at other positions had yields ranging from 71% to 92% (3i, 3j, 3k, 3m). Compared to benzaldehydes with electron-withdrawing groups, benzaldehydes with methoxy groups or no substituents showed lower yields, with 3n at 78% and 3o at 63%. Furthermore, we investigated aldehydes with special structures, such as phenylpropanal (3p) with an aliphatic chain, and found that yields as high as 93% could be obtained, indicating that this method also has the potential to achieve acetalization reactions of aliphatic aldehydes. In the conventional protonic acid-catalyzed acetal reaction of cinnamaldehyde (3q), the double bond typically undergoes addition, but this method yields 67% yield, effectively avoiding the problem of simultaneous reaction of the double bond. Similarly, 4-ethynylbenzaldehyde achieves a yield of 98% (3r). Larger acetals like 4-biphenylbenzaldehyde also achieve a yield of 80% (3s). Notably, tert-butyloxycarbonyl-protected 4-aminobenzaldehyde (3t) also exhibits a very high acetalization yield (89%); however, this substrate undergoes decomposition under conventional acid catalysis, removing the protecting group and exposing a highly reactive amino group that is easily destroyed, hindering subsequent reactions. We also used 4-acetylbenzaldehyde as a substrate (3u) to investigate the selectivity of the reaction for ketone carbonyl and aldehyde groups, finding that only the aldehyde group reacted, while the ketone carbonyl group remained unreacted, with a yield of 73%.

[0083] II. A tandem reaction of transesterification and acetal reaction catalyzed by bovine trypsinogen.

[0084]

[0085] Bovine trypsinogen catalyzes the transesterification reaction of ethylene ester with a methanol-containing solution, releasing vinyl alcohol. Vinyl alcohol is unstable and spontaneously converts to acetaldehyde in situ. Acetaldehyde further reacts with a methanol-containing solution under the catalysis of bovine trypsinogen to form acetal, yielding 1,1-dimethoxyethane.

[0086] The ethylene ester includes, but is not limited to, vinyl acetate, vinyl propionate, vinyl butyrate, or vinyl hexanoate; the methanol-containing solution is a pure methanol solution, a methanol-ethanol mixture, a methanol-n-propanol mixture, a methanol-isopropanol mixture, a methanol-n-butanol mixture, or a methanol-isobutanol mixture; preferably, the reaction temperature is 37℃~60℃, and the reaction time is 24~144 h.

[0087] 1. Preparation of 1,1-dimethoxyethane using bovine trypsinogen as a catalyst.

[0088] Example 28

[0089] like Figure 1 As shown in (B), 50 μL of vinyl acetate, 250 μL of methanol, and 1.0 mg of bovine trypsinogen were added to a sealed reaction tube, mixed thoroughly, and incubated in a shaker at 200 rpm and 50 °C for 72 h to obtain 1,1-dimethoxyethane. The yield was determined to be 27% by gas chromatography.

[0090] The compounds were characterized by ¹H NMR spectroscopy and gas chromatography-mass spectrometry (GC-MS). ¹H NMR detection conditions: Agilent DD2-500 MHz NMR spectrometer, CDCl3 as solvent (d = 7.26 ppm). GC-MS detection conditions: Agilent 7890-5977 GC-MS instrument, EI ion source, ion source temperature 230℃, quadrupole temperature 150℃, HP-5MS column (30 m × 0.25 mm × 0.25 μm), helium as carrier gas, column flow rate 1.0 mL / min, and programmed temperature ramp (initial 40℃ held for 2 min, then ramped to 150℃ at a rate of 4℃ / min and held for 2 min). The gas chromatography detection conditions were as follows: a GC112N gas chromatograph from Shanghai Instrument & Electronics Analytical Instrument Co., Ltd., a TVOC-specific column (50 m × 0.32 mm × 0.25 μm), nitrogen as the carrier gas, a column flow rate of 2.0 mL / min, a vaporization chamber temperature of 230℃, a detector temperature of 250℃, and a programmed temperature ramp (initially 60℃ and held for 2 min, then ramped to 200℃ at a rate of 5℃ / min and held for 10 min). The 1H NMR results are shown below. Figure 5 As shown, the gas chromatography-mass spectrometry (GC-MS) detection results are as follows: Figure 6 and Figure 7 As shown, the product structure was determined to be 1,1-dimethoxyethane.

[0091] 2. Investigation of reaction conditions.

[0092] 1) The effect of different ethylene esters on the tandem reaction of transesterification and acetal reaction.

[0093] Table 5. Effects of different ethylene esters on the tandem reactions of transesterification and acetalization.

[0094]

[0095] As shown in Table 5, referring to Example 28, vinyl acetate was replaced with vinyl propionate, vinyl butyrate, vinyl hexanoate, vinyl crotonate, vinyl pentanoate, and vinyl benzoate, respectively, to prepare Examples 29 to 31 and Comparative Examples 9 to 11.

[0096] Comparing Examples 29-31 and Comparative Examples 9-11, it was found that when using vinyl acetate, which has the smallest structure, a yield of up to 27% could be obtained. Conversely, as the ester moiety gradually increased, the yield decreased from 20% to trace amounts. Due to the fine spatial structure and active regions of biomacromolecules, there is a significant selectivity for size differences in small organic molecules. Based on these results, vinyl acetate was determined to be the most suitable substrate.

[0097] 2) The effect of different catalysts on the tandem reaction of transesterification and acetal reaction.

[0098] Table 6. Effects of different catalysts on the tandem reaction of transesterification and acetalization.

[0099]

[0100] As shown in Table 6, referring to Example 28, bovine trypsinogen was replaced with bovine trypsin, bovine serum albumin and no catalyst were added respectively to prepare Comparative Examples 12 to 14.

[0101] Comparative Examples 28 and 12-14 show that replacing bovine trypsinogen with bovine trypsin or bovine serum albumin, or not using a catalyst, results in significantly lower yields of the tandem transesterification and acetal reactions compared to bovine trypsinogen, or even no reaction at all. These results demonstrate that bovine trypsinogen can accept different non-natural substrates and simultaneously exert two activities to catalyze the successful preparation of 1,1-dimethoxyethane in this tandem reaction.

[0102] 3) The effect of the raw material volume ratio on the tandem reaction of transesterification and acetal reaction.

[0103] Table 7. Effect of feedstock volume ratio on the tandem reaction of transesterification and acetalization.

[0104]

[0105] Referring to Example 28, Examples 32-35, Comparative Examples 15 and 16 were prepared according to the volume ratio of vinyl acetate to methanol shown in Table 7.

[0106] According to the reaction formula, the reaction can only proceed smoothly when the molar ratio of vinyl acetate to methanol is 1:3. Comparing Examples 28, 32-35, and Comparative Examples 15 and 16, the results showed that the yield increased as the volume of methanol gradually increased, i.e., the volume of vinyl acetate gradually decreased. When the volume of methanol was in large excess (>5:1), the yield could reach ≥30%, which also proved that methanol played multiple roles in this reaction system: firstly, as a raw material for the first step of transesterification reaction; secondly, as a raw material for the second step of acetal reaction; and thirdly, as a reaction medium condition. This also helps to reduce the introduction of other reagents and reduce the difficulty of post-processing. Therefore, 25 μL of vinyl acetate and 275 μL of methanol (Example 33) were selected to continue optimizing the subsequent conditions.

[0107] 4) The effect of mixed alcohol system ratio on the tandem reaction of transesterification and acetal reaction.

[0108] Table 8. Effect of mixed alcohol system ratio on the tandem reaction of transesterification and acetal reaction

[0109]

[0110] As shown in Table 8, with reference to Example 28, the methanol content was adjusted to prepare Examples 36 to 42, as well as Comparative Examples 17 and 18.

[0111] Comparing Examples 28, 36-42, 17, and 18, the results show that adding a certain volume of other alcohols does affect the yield, but the effect is not significant. Furthermore, taking the addition of ethanol as an example, the effect of the addition ratio on the reaction was also investigated. Experimental data show that as the volume ratio of ethanol increases, the yield exhibits a significant decreasing trend. Since adding other alcohols does not significantly improve the reaction effect, but instead introduces an additional reagent and provides the possibility of side reactions, considering all factors, methanol alone will remain the most suitable reagent for subsequent experiments.

[0112] 5) The effect of bovine trypsinogen dosage on the tandem reaction of transesterification and acetal reaction.

[0113] Table 9. Effect of bovine trypsinogen dosage on the tandem reaction of transesterification and acetalization.

[0114]

[0115] As shown in Table 9, referring to Example 33, the amount of bovine trypsinogen was adjusted to prepare Examples 43 to 45 respectively.

[0116] Comparing Example 33 (parallel experiment 1) and Examples 43-45, it was found that 1.0-2.0 mg of bovine trypsinogen was sufficient to maximize the reaction yield, and reducing or increasing the amount of catalyst decreased the yield. Therefore, 1.0 mg of catalyst was determined to be the optimal amount for this reaction.

[0117] 6) The effect of reaction temperature and time on the tandem reaction of transesterification and acetalization.

[0118] Table 10 Effects of reaction temperature and time on the tandem reaction of transesterification and acetalization

[0119]

[0120] As shown in Table 10, with reference to Example 33, the reaction time (12~144 h) and temperature (37℃, 50℃ and 60℃) were adjusted to prepare Examples 46~64 and Comparative Example 19, respectively.

[0121] Comparing Example 33 (parallel experiment 2), Examples 46-64, and Comparative Example 19, the results show that when reacting at 37°C, the yield continuously increases with time, reaching a maximum of 32% at 144 h. It is foreseeable that the yield will increase further with continued extension of the reaction time. When reacting at 50°C, the yield shows a similar trend, but the upward trend gradually slows down after 96 h, reaching a maximum of 50%. When reacting at 60°C, the yield is significantly higher than the previous two temperature conditions within the same time frame, reaching a maximum of 63% at 96 h. Further extension of the reaction time does not significantly change the yield. Therefore, reacting at 60°C for 96 h is the optimal condition for preparing 1,1-dimethoxyethane using this method.

[0122] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for utilizing bovine trypsinogen to catalyze an acetal reaction, characterized by, The O, O-acetal is obtained by catalyzing the acetalization of aromatic aldehyde with a methanol-containing solution by using bovine trypsinogen; Or, the ethenol is obtained by catalyzing the transesterification of ethylene ester with a methanol-containing solution by using bovine trypsinogen, and the ethenol is spontaneously converted into acetaldehyde in situ; the acetaldehyde is further catalyzed by bovine trypsinogen to react with a methanol-containing solution to obtain 1, 1-dimethoxyethane; the ethylene ester is vinyl acetate, vinyl propionate, vinyl butyrate or vinyl hexanoate; The volume fraction of methanol in the methanol-containing solution is greater than 70%.

2. The method of claim 1, wherein the catalytic acetal reaction is catalyzed by bovine trypsinogen. In the reaction for preparing the O, O-acetal, the aromatic aldehyde is benzaldehyde.

3. The method of claim 1, wherein the catalytic acetal reaction is catalyzed by bovine trypsinogen. In the reaction for preparing the O, O-acetal, the reaction temperature is 37-60 DEG C, and the reaction time is 1-12 h.

4. The method of claim 1, wherein the catalytic acetal reaction is catalyzed by bovine trypsinogen. In the reaction for preparing the 1, 1-dimethoxyethane, the methanol-containing solution is pure methanol solution, methanol-ethanol mixed solution, methanol-n-propanol mixed solution, methanol-isopropanol mixed solution, methanol-n-butanol mixed solution or methanol-isobutanol mixed solution.

5. The method of claim 1, wherein the catalytic acetal reaction is catalyzed by bovine trypsinogen. In the reaction for preparing the 1, 1-dimethoxyethane, the volume of the methanol-containing solution is greater than that of the ethylene ester.

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