A method for detecting the synthesis of LNT II by enzyme catalysis process

Abstract

The application belongs to the field of chemical analysis, and particularly relates to a method for detecting LNT II synthesized by an enzyme catalysis process, comprising the following process: in the process of synthesizing an intermediate and / or end product of LNT II by an enzyme catalysis process, the reaction solution after inactivation and protein removal is detected by HPLC-UV-RI, and the detection result is quantitatively analyzed; the chromatographic condition of the HPLC-UV-RI detection is as follows: a HILIC column of a polymer matrix, a column temperature of 50-60 DEG C, a mobile phase A of acetonitrile, a mobile phase B of a potassium phosphate dibasic aqueous solution, a concentration of the potassium phosphate dibasic aqueous solution of 40-60 mmol / L, a pH value of 6.5-7.5, and a volume ratio of the mobile phase A to the mobile phase B of (65-75):(35-25). The method provided by the application can effectively separate and accurately quantify the substrate, intermediate, end product and the like in the reaction solution in the process of synthesizing LNT II by an enzyme catalysis process.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis, and particularly relates to a central control detection method for the synthesis of LNT II by enzyme catalysis. Background Technology

[0002] Human milk oligosaccharides (HMOs) are a class of complex, non-digestible carbohydrates found in human milk. As the third largest solid component of breast milk after lactose and fat, HMOs are important bioactive factors that play a unique role in infant growth and development, significantly impacting the growth, development, and health of newborns. They are used in infant formula to provide unique health benefits that cannot be provided by regular milk powder, and the addition of HMOs to infant formula has become a significant trend in the premium formula industry.

[0003] HMOs are diverse, mainly including lactose-N-neotetrasaccharides, lactose-N-tetrasaccharides, 3-fucosylvose, 2'-fucosylvose, 6'-sialyllactose, and 3'-sialyllactose. Lactose-N-trisaccharide II (LNT II) is an important core structural unit in HMOs and can be used as a precursor to produce lactose-N-neotetrasaccharides (LNnT) and lactose-N-tetrasaccharides (LNT). Traditionally, LNTII is synthesized through chemical methods, which require multiple steps such as activation, protection, and deprotection to achieve stereoselectivity and regioselectivity in glycosidic bond synthesis. In contrast, enzymatic catalysis has attracted widespread attention in recent years due to its mild aqueous environment and high stereoselectivity and regioselectivity for unprotected substrates.

[0004] The main process of LNT II synthesis via enzymatic catalysis includes: Step 1 uses acetylglucosamine (GlcNAc) and adenosine triphosphate (ATP) as substrates, catalyzed by acetylglucosamine kinase to generate intermediate 1 (acetylglucosamine-1-phosphate (GlcNAc-1-P)); Step 2 uses intermediate 1 and uridine triphosphate (UTP) as substrates, catalyzed by acetylglucosamine-1-P uridine transferase to generate intermediate 2 (uridine 5'-bisphosphate-N-acetylglucosamine (UDP-GlcNAc)); Step 3 uses lactose and intermediate 2 as substrates, catalyzed by acetylglucosamine transferase to synthesize LNT II. Quantitative detection of the substrates, intermediates, and final products involved in the above processes is of great significance for quality control of LNT II synthesis via enzymatic catalysis. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a mid-control detection method for the synthesis of LNT II by enzyme catalysis. This method can effectively separate and accurately quantify the substrate, intermediates, and final products in the reaction solution during the synthesis of LNT II by enzyme catalysis, thereby effectively controlling each step of the reaction and ensuring the conversion rate and yield of each step in the synthesis process.

[0006] This invention provides a method for the intermediate-control detection of enzyme-catalyzed synthesis of LNTII, comprising the following steps:

[0007] During the enzyme-catalyzed synthesis of intermediates and / or final products of LNTⅡ, the reaction solution after inactivation and protein removal was detected by HPLC-UV-RI, and the detection results were quantitatively analyzed.

[0008] The chromatographic conditions for HPLC-UV-RI detection are as follows: the chromatographic column is a polymer-based HILIC column, the column temperature is 50-60℃, mobile phase A is acetonitrile, mobile phase B is a dipotassium hydrogen phosphate aqueous solution with a concentration of 40-60 mmol / L, the pH value of the dipotassium hydrogen phosphate aqueous solution is 6.5-7.5, and the volume ratio of mobile phase A to mobile phase B is (65-75):(35-25).

[0009] Preferably, the specific synthetic route for the enzyme-catalyzed synthesis of LNTⅡ is as follows:

[0010] S1) Using acetylglucosamine and adenosine triphosphate as substrates, the first intermediate is generated by acetylglucosamine kinase catalysis. The first intermediate is acetylglucosamine-1-phosphate.

[0011] S2) Using the first intermediate and uridine triphosphate as substrates, a second intermediate is generated by catalysis of acetylglucosamine-1-P uridine transferase. The second intermediate is uridine 5'-bisphosphate-N-acetylglucosamine.

[0012] S3) Using the second intermediate and lactose as substrates, the final product lactose-N-trisaccharide II is synthesized by catalysis of acetylglucosamine transferase.

[0013] Preferably, during the HPLC-UV-RI detection process, the substances measured include one or more of acetylglucosamine, lactose, acetylglucosamine-1-phosphate, adenosine triphosphate, adenosine monophosphate, adenosine diphosphate, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine 5'-bisphosphate-N-acetylglucosamine, and lactose-N-trisaccharide II.

[0014] Preferably, the column temperature is 55°C.

[0015] Preferably, the concentration of the dipotassium hydrogen phosphate aqueous solution is 50 mmol / L, and the pH value of the dipotassium hydrogen phosphate aqueous solution is 7.

[0016] Preferably, the volume ratio of mobile phase A to mobile phase B is 70:30.

[0017] Preferably, the specific method for inactivating and removing proteins is as follows: mixing the reaction solution with an acetonitrile aqueous solution, followed by filtration.

[0018] Preferably, the concentration of the acetonitrile aqueous solution is 65-75 vol%; therefore, the pore size of the filter membrane is 0.15-0.3 μm.

[0019] Preferably, the wavelength of the ultraviolet detector used in the HPLC-UV-RI detection process is 205-215 nm.

[0020] Preferably, during the HPLC-UV-RI detection process, the temperature of the differential refractive index detector used is 35–45°C.

[0021] Compared with existing technologies, this invention provides a mid-control detection method for the enzyme-catalyzed synthesis of LNT II, ​​comprising the following steps: during the enzyme-catalyzed synthesis of intermediates and / or final products of LNT II, ​​the reaction solution after inactivation and protein removal is analyzed by HPLC-UV-RI, and the detection results are quantitatively analyzed; the chromatographic conditions for HPLC-UV-RI detection are: a polymer-based HILIC column, a column temperature of 50-60℃, mobile phase A being acetonitrile, mobile phase B being a dipotassium hydrogen phosphate aqueous solution with a concentration of 40-60 mmol / L, a pH of 6.5-7.5, and a volume ratio of mobile phase A to mobile phase B of (65-75):(35-25). The method provided by this invention, by inactivating and removing proteins from the reaction solution and performing HPLC-UV-RI coupled detection analysis on the treated reaction solution under appropriate chromatographic conditions, achieves effective separation and accurate quantification of substrates, intermediates, and final products in the reaction solution during the enzyme-catalyzed synthesis of LNT II. The detection method provided by this invention has high specificity, high sensitivity, and good linear correlation. The sample pretreatment process is simple and quick. This method can accurately detect the content of 11 compounds (GlcNAc, lactose, GlcNAc-1-P, UDP-GlcNAc, ATP, adenosine monophosphate (AMP), adenosine diphosphate (ADP), UTP, uridine diphosphate (UDP), uridine monophosphate (UMP), and LNT II) involved in the enzyme-catalyzed synthesis of LNT II. This provides data support for the process monitoring of enzyme-catalyzed synthesis of LNT II, ​​ensuring that each step of the synthesis process can be effectively controlled and guaranteeing the conversion rate and yield of each step. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 This is a spectrum of UTP / UDP / UMP separated by a C18 column provided in an embodiment of the present invention;

[0024] Figure 2 This is a spectrum of GlcNAc separated at a column temperature of 40°C, provided in an embodiment of the present invention.

[0025] Figure 3 This is a spectrum of GlcNAc separated at a column temperature of 45°C, provided in an embodiment of the present invention.

[0026] Figure 4 This is a spectrum of GlcNAc separated at a column temperature of 50°C, provided in an embodiment of the present invention.

[0027] Figure 5 This is a spectrum of GlcNAc separated at a column temperature of 55°C, provided in an embodiment of the present invention.

[0028] Figure 6 This is a control spectrum of the first step reaction of the enzyme-catalyzed synthesis of LNT II provided in the embodiments of the present invention;

[0029] Figure 7 This is a control spectrum of the second step reaction of the enzyme-catalyzed synthesis of LNT II provided in the embodiments of the present invention;

[0030] Figure 8 This is a mid-range spectrum (UV spectrum) of the third step reaction of the enzyme-catalyzed synthesis of LNT II provided in the embodiments of the present invention;

[0031] Figure 9 This is a control spectrum (RI diagram) of the third step reaction of the enzyme-catalyzed synthesis of LNT II provided in the embodiments of the present invention;

[0032] Figure 10 This is the specificity assessment - UV spectrum provided in the embodiments of the present invention;

[0033] Figure 11 This is the specificity assessment-RI spectrum provided in the embodiments of the present invention;

[0034] Figure 12 This is a lactose linear graph provided in an embodiment of the present invention;

[0035] Figure 13 This is the GlcNAc linear graph provided in the embodiments of the present invention;

[0036] Figure 14 This is the LNT II linear graph provided in the embodiments of the present invention;

[0037] Figure 15 This is a UDP-GlcNAc linear graph provided in an embodiment of the present invention;

[0038] Figure 16 This is the GlcNAc-1-P linear graph provided in the embodiments of the present invention;

[0039] Figure 17 This is a UMP linear graph provided in an embodiment of the present invention;

[0040] Figure 18 This is the AMP linear graph provided in the embodiments of the present invention;

[0041] Figure 19This is a UDP linear graph provided in an embodiment of the present invention;

[0042] Figure 20 This is the ADP linear graph provided in the embodiments of the present invention;

[0043] Figure 21 This is a UTP linear graph provided in an embodiment of the present invention;

[0044] Figure 22 This is the ATP linear graph provided in the embodiments of the present invention;

[0045] Figure 23 This is the limit of quantitation chart of UDP-GlcNAc / UMP / AMP / ADP provided in the embodiments of the present invention;

[0046] Figure 24 This is a UDP / ATP quantitation limit diagram provided in an embodiment of the present invention;

[0047] Figure 25 This is a limit of quantitation (LOQ) chart of GlcNAc provided in an embodiment of the present invention;

[0048] Figure 26 The limit of quantitation (LOQ) map of GlcNAc-1-P / UTP provided in this embodiment of the invention;

[0049] Figure 27 The limit of quantitation (LOQ) chart of LNTⅡ provided in this embodiment of the invention;

[0050] Figure 28 The limit of quantification (RI) chart for lactose provided in this embodiment of the invention. Detailed Implementation

[0051] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] This invention provides a method for the intermediate-control detection of enzyme-catalyzed synthesis of LNTII, comprising the following steps:

[0053] During the enzyme-catalyzed synthesis of intermediates and / or final products of LNTⅡ, the reaction solution after inactivation and protein removal was detected by HPLC (high performance liquid chromatography)-UV (ultraviolet spectrophotometry)-RI (differential refractive index detector), and the detection results were quantitatively analyzed.

[0054] In the detection method provided by this invention, the preferred synthetic route for the enzyme-catalyzed synthesis of LNTⅡ is as follows:

[0055] S1) uses GlcNAc and ATP as substrates to generate the first intermediate (GlcNAc-1-P) through acetylglucosamine kinase catalysis;

[0056] S2) Using the first intermediate and UTP as substrates, the second intermediate (UDP-GlcNAc) is generated by catalysis of acetylglucosamine-1-P uridine transferase.

[0057] S3) Using the second intermediate and lactose as substrates, the final product LNTⅡ is synthesized by catalysis of acetylglucosamine transferase.

[0058] In the detection method provided by this invention, the preferred method for inactivation and protein removal is to mix the reaction solution with an acetonitrile aqueous solution, followed by filtration. The concentration of the acetonitrile aqueous solution is preferably 65–75 vol%, more preferably 70 vol%; therefore, the pore size of the filter membrane is preferably 0.15–3 μm, more preferably 0.22 μm.

[0059] In the detection method provided by the present invention, during the HPLC-UV-RI detection process, the selected chromatographic column is a polymer-based HILIC column, preferably a HILIC pak VG-504E; the column temperature is 50-60℃, more preferably 55℃.

[0060] In the detection method provided by this invention, during the HPLC-UV-RI detection process, a dipotassium hydrogen phosphate-acetonitrile system is used as the mobile phase. More specifically, acetonitrile is used as mobile phase A, and an aqueous solution of dipotassium hydrogen phosphate is used as mobile phase B. The concentration of the dipotassium hydrogen phosphate aqueous solution is 40–60 mmol / L, preferably 50 mmol / L; the pH value of the dipotassium hydrogen phosphate aqueous solution is 6.5–7.5, preferably 7; the pH value of the dipotassium hydrogen phosphate aqueous solution is adjusted by phosphoric acid; the volume ratio of mobile phase A to mobile phase B is (65–75):(35–25), preferably 70:30; the flow rate of the mobile phase in the chromatographic column is preferably 1–2 mL / min, more preferably 1.5 mL / min.

[0061] In the detection method provided by the present invention, during the HPLC-UV-RI detection process, the sample plate temperature of the high-performance liquid chromatograph is preferably 10-20°C, more preferably 15°C.

[0062] In the detection method provided by the present invention, during the HPLC-UV-RI detection process, the wavelength of the ultraviolet detector used is preferably 205-215 nm, more preferably 210 nm.

[0063] In the detection method provided by the present invention, during the HPLC-UV-RI detection process, the temperature of the differential refractive index detector used is preferably 35-45°C, more preferably 40°C.

[0064] In the detection method provided by the present invention, during the HPLC-UV-RI detection process, the injection volume is preferably 5-15 μL, more preferably 10 μL.

[0065] In the detection method provided by this invention, during the HPLC-UV-RI detection process, the substances measured preferably include one or more of GlcNAc, lactose, GlcNAc-1-P, ATP, AMP, ADP, UTP, UDP, UMP, UDP-GlcNAc, and LNT II. More specifically, for step S1) of synthesizing the first intermediate, the substances measured in the reaction solution include GlcNAc, GlcNAc-1-P, ATP, AMP, and ADP; for step S2) of synthesizing the second intermediate, the substances measured in the reaction solution include UDP-GlcNAc, GlcNAc, GlcNAc-1-P, ATP, AMP, ADP, UTP, UDP, and UMP; for step S3) of synthesizing the final product, the substances measured in the reaction solution include UDP-GlcNAc, GlcNAc, GlcNAc-1-P, ATP, AMP, ADP, UTP, UDP, UMP, lactose, and LNT II.

[0066] In the detection method provided by the present invention, it is preferable to further perform HPLC-UV-RI detection on the blank group solution and the reference solution. The blank group solution is a filtered aqueous acetonitrile solution, the concentration of which is the same as that used in the inactivation treatment of the reaction solution, and the pore size of the filter membrane is the same as that used in the protein removal treatment of the reaction solution. The reference solution is a standard solution of known concentration that has undergone inactivation and protein removal, and the specific type of the standard is determined based on the composition of the reaction solution to be measured.

[0067] In the detection method provided by the present invention, the quantitative analysis is preferably performed by using the external standard method to quantitatively calculate the detection results.

[0068] The method provided by this invention achieves effective separation and accurate quantification of substrates, intermediates, and final products in the enzyme-catalyzed synthesis of LNT II by inactivating and deproteinizing the reaction solution and performing HPLC-UV-RI coupled analysis under suitable chromatographic conditions. The detection method provided by this invention is highly specific, sensitive, and linearly correlated, and the sample pretreatment process is simple and rapid. This method can accurately detect the content of 11 compounds (GlcNAc, lactose, GlcNAc-1-P, UDP-GlcNAc, ATP, adenosine monophosphate (AMP), adenosine diphosphate (ADP), UTP, uridine diphosphate (UDP), uridine monophosphate (UMP), and LNT II) involved in the enzyme-catalyzed synthesis of LNT II, ​​thereby providing data support for process monitoring of the enzyme-catalyzed synthesis of LNT II, ​​ensuring effective control of each reaction step in the synthesis process, and guaranteeing the conversion rate and yield of each reaction step.

[0069] The relevant technical mechanism and key points of the detection method of this invention are as follows:

[0070] (1) The method of this invention is used for process control of enzyme-catalyzed synthesis of LNT II. It mainly involves the detection and monitoring of 11 compounds (GlcNAc, lactose, GlcNAc-1-P, UDP-GlcNAc, ATP, AMP, ADP, UTP, UDP, UMP, and LNT II) involved in the enzyme-catalyzed synthesis of LNT II. The logP values ​​of these 11 compounds, calculated using Chemdraw software, were -2.82, -4.12, -2.85, -4.56, -3.22, -3.88, -4.60, -3.95, -4.14, -3.74, and -3.51, respectively. It can be seen that the logP values ​​(octanol / water partition coefficient) of these 11 compounds are all <0, indicating that the compounds are weakly hydrophobic and highly polar. Using conventional hydrophobic separation mechanisms with C18, C8, and C4 columns for separation and elution results in weak compound retention and poor peak resolution (see...). Figure 1 This invention uses a polymer-based HILIC column, which utilizes a hydrophilic interaction mechanism to enhance the retention of target analytes. Compared with silica-based HILIC columns, polymer-based HILIC columns have better chemical stability, longer lifespan, and less change in separation efficiency over time.

[0071] (2) Because the GlcNAc structure contains an aldehyde group, there is tautomerism between the aldehyde and enol forms in solution, leading to abnormal chromatographic peak shapes (see...). Figures 2-4 Increasing the column temperature is beneficial for improving peak shape. Under the conditions of satisfying both peak shape and column tolerance, a column temperature of 55℃ was determined to be the optimal temperature (see...). Figure 5 Simultaneously, different buffer salt concentrations, different pH values, and different mobile phase ratios were explored to determine the optimal mobile phase salt phase as 50 mM dipotassium hydrogen phosphate (pH = 7.0) and the optimal elution ratio as acetonitrile:salt phase = 70:30. Under these conditions, the peak shapes of the 11 compounds were good, and the resolution of each peak met the detection requirements.

[0072] (3) Lactose has no chromophores in its structure and does not absorb under a UV detector. This invention uses HPLC-UV-RI to analyze and detect the contents of GlcNAc, lactose, GlcNAc-1-P, UDP-GlcNAc, ATP, AMP, ADP, UTP, UDP, UMP, and LNT II during the reaction.

[0073] (4) Since the reaction solution of the biological enzyme method contains a large amount of enzyme protein, which has biological activity, it needs to be inactivated and protein removed after sampling. Otherwise, it will affect the judgment of the detection results and cause the liquid chromatography system to malfunction. Therefore, the present invention preferably uses 70 vol% acetonitrile aqueous solution for reaction solution inactivation treatment, which can effectively inactivate enzyme protein and precipitate protein. It is also preferred to use a filter membrane with a pore size of 0.22 μm to remove protein and protect the residual protein from the influence of the chromatography system.

[0074] For clarity, the following examples will be used to provide a detailed description.

[0075] Example 1

[0076] This embodiment provides a method for the first step of the enzyme-catalyzed synthesis of LNTII, which involves the detection of GlcNAc, GlcNAc-1-P, ATP, AMP, and ADP, and the calculation of the content of each substance and the conversion rate of the reaction.

[0077] The method includes the following steps:

[0078] ① Testing conditions:

[0079] Chromatographic conditions: Mobile phase A was acetonitrile, mobile phase B was 50 mmol / L dipotassium hydrogen phosphate aqueous solution (pH adjusted to 7.0 with phosphoric acid), the volume ratio of mobile phase A to mobile phase B was 70:30, the flow rate of the mobile phase in the column was 1.5 mL / min, the column was HILICpak VG-504E (length 250 mm, inner diameter 4.6 mm, particle size 5 μm), the injection volume was 10 μL, the column temperature was 55 ℃, the sample tray temperature was 15 ℃, and the run time was 35 min.

[0080] Ultraviolet detector: wavelength 210nm;

[0081] Differential detector: temperature 40℃.

[0082] ② Blank solution: 70 vol% acetonitrile aqueous solution, filtered through a 0.22 μm organic phase filter membrane, and the filtrate is used for analysis.

[0083] ③ Sample solution preparation:

[0084] Accurately measure 1 mL of the first step reaction solution, place it in a 10 mL volumetric flask, dilute to the mark with 70 vol% acetonitrile aqueous solution, shake well, filter through a 0.22 μm organic phase filter membrane, and take the filtrate for testing.

[0085] ④ Preparation of reference solution: Accurately weigh 25 mg each of GlcNAc, GlcNAc-1-P, ATP, AMP, and ADP and place them in a 50 mL volumetric flask. Add 70 vol% acetonitrile aqueous solution to dissolve and dilute to the mark. Shake well and filter through a 0.22 μm organic phase filter membrane. Take the filtrate for testing.

[0086] ⑤ Determination: The test solutions in ②, ③, and ④ were analyzed by HPLC-UV-RI using the detection conditions in ①, and the chromatograms were obtained (see...). Figure 6 The contents of GlcNAc, GlcNAc-1-P, ATP, AMP, and ADP were calculated using the external standard method. The results are shown in Table 1.

[0087] Table 1. Quantitative analysis results of the first step reaction solution

[0088]

[0089] Example 1 shows that the method of the present invention can effectively and accurately monitor the content and conversion rate of each substance in the first step of the enzyme catalysis process.

[0090] Example 2

[0091] This embodiment provides a second-step control detection method for the enzyme-catalyzed synthesis of LNTII. The substances to be measured are UDP-GlcNAc, GlcNAc, GlcNAc-1-P, ATP, AMP, ADP, UTP, UDP, and UMP. The content of each substance and the conversion rate of the reaction are calculated.

[0092] The method includes the following steps:

[0093] ① Testing conditions:

[0094] Chromatographic conditions: Mobile phase A was acetonitrile, mobile phase B was 50 mmol / L dipotassium hydrogen phosphate aqueous solution (pH adjusted to 7.0 with phosphoric acid), the volume ratio of mobile phase A to mobile phase B was 70:30, the flow rate of the mobile phase in the column was 1.5 mL / min, the column was HILICpak VG-504E (length 250 mm, inner diameter 4.6 mm, particle size 5 μm), the injection volume was 10 μL, the column temperature was 55 ℃, the sample tray temperature was 15 ℃, and the run time was 35 min.

[0095] Ultraviolet detector: wavelength 210nm;

[0096] Differential detector: temperature 40℃.

[0097] ② Blank solution: 70 vol% acetonitrile aqueous solution, filtered through a 0.22 μm organic phase filter membrane, and the filtrate is used for analysis.

[0098] ③ Sample solution preparation:

[0099] Accurately measure 1 mL of the reaction solution from the second step, place it in a 10 mL volumetric flask, dilute to the mark with 70 vol% acetonitrile aqueous solution, shake well, filter through a 0.22 μm organic phase filter membrane, and take the filtrate for testing.

[0100] ④ Preparation of reference solution: Accurately weigh 25 mg each of UDP-GlcNAc, GlcNAc, GlcNAc-1-P, ATP, AMP, ADP, UTP, UDP, and UMP and place them in a 50 mL volumetric flask. Add 70 vol% acetonitrile aqueous solution to dissolve and dilute to the mark. Shake well and filter through a 0.22 μm organic phase filter membrane. Take the filtrate for testing.

[0101] ⑤ Determination: The test solutions in ②, ③, and ④ were analyzed by HPLC-UV-RI using the detection conditions in ①, and the chromatograms were obtained (see...). Figure 7 The contents of UDP-GlcNAc, GlcNAc, GlcNAc-1-P, ATP, AMP, ADP, UTP, UDP, and UMP were calculated using the external standard method. The results are shown in Table 2.

[0102] Table 2 Quantitative analysis results of the reaction solution in the second step

[0103]

[0104] Example 2 shows that the method of the present invention can effectively and accurately monitor the content and conversion rate of each substance in the second step of the enzyme catalysis process.

[0105] Example 3

[0106] This embodiment provides a third-step control detection method for the enzyme-catalyzed synthesis of LNTII. The substances to be measured are UDP-GlcNAc, GlcNAc, GlcNAc-1-P, ATP, AMP, ADP, UTP, UDP, UMP, lactose, and LNTII. The content of each substance and the conversion rate of the reaction are calculated.

[0107] The method includes the following steps:

[0108] ① Testing conditions:

[0109] Chromatographic conditions: Mobile phase A was acetonitrile, mobile phase B was 50 mmol / L dipotassium hydrogen phosphate aqueous solution (pH adjusted to 7.0 with phosphoric acid), the volume ratio of mobile phase A to mobile phase B was 70:30, the flow rate of the mobile phase in the column was 1.5 mL / min, the column was HILICpak VG-504E (length 250 mm, inner diameter 4.6 mm, particle size 5 μm), the injection volume was 10 μL, the column temperature was 55 ℃, the sample tray temperature was 15 ℃, and the run time was 35 min.

[0110] Ultraviolet detector: wavelength 210nm;

[0111] Differential detector: temperature 40℃.

[0112] ② Blank solution: 70 vol% acetonitrile aqueous solution, filtered through a 0.22 μm organic phase filter membrane, and the filtrate is used for analysis.

[0113] ③ Sample solution preparation:

[0114] Accurately measure 1 mL of the reaction solution from step 3, place it in a 10 mL volumetric flask, dilute to the mark with 70 vol% acetonitrile aqueous solution, shake well, filter through a 0.22 μm organic phase filter membrane, and take the filtrate for testing.

[0115] ④ Preparation of reference solution: Accurately weigh 25 mg each of UDP-GlcNAc, GlcNAc, GlcNAc-1-P, ATP, AMP, ADP, UTP, UDP, UMP, lactose, and LNTⅡ into a 50 mL volumetric flask, add 70 vol% acetonitrile aqueous solution to dissolve and dilute to the mark, shake well, filter through a 0.22 μm organic phase filter membrane, and take the filtrate for detection.

[0116] ⑤ Determination: The test solutions in ②, ③, and ④ were analyzed by HPLC-UV-RI using the detection conditions in ①, and the chromatograms were obtained (see...). Figure 8 , Figure 9 ); UV chromatogram (see Figure 8The contents of UDP-GlcNAc, GlcNAc, GlcNAc-1-P, ATP, AMP, ADP, UTP, UDP, UMP, and LNTⅡ were calculated using the external standard method; the RI chromatogram (see RI chromatogram) Figure 9 The lactose content was calculated using the external standard method. The test results are shown in Table 3.

[0117] Table 3. Quantitative analysis results of the reaction solution in step 3.

[0118] Quantitative substances Content (mg / mL) GlcNAc 1.1 GlcNAc-1-P 0.4 ATP 3.1 AMP 9.5 ADP 7.0 UDP-GlcNAc 2.4 UTP 5.0 UDP 9.4 UMP 1.1 LNTⅡ 22.9 lactose 3.1 Reaction conversion rate (%) 84.0％

[0119] Example 3 shows that the method of the present invention can effectively and accurately monitor the content and conversion rate of each substance in the third step of the enzyme catalysis process.

[0120] Example 4

[0121] This embodiment conducts an experimental methodological review of the LNTⅡ central control detection method provided by the present invention, and the results are as follows:

[0122] ① Specificity examination

[0123] A mixed solution of GlcNAc, lactose, GlcNAc-1-P, UDP-GlcNAc, ATP, AMP, ADP, UTP, UDP, UMP, and LNTII was prepared, along with a diluent. Analysis was performed using the detection method provided in Example 3. The results showed that the diluent did not interfere with the peak positions of LNTII and the other substances. The resolution of all 11 compounds was greater than 1.5, meeting the baseline separation requirements, indicating that the detection method of this invention has strong specificity. The peak elution times and resolutions are shown in Table 4 (see chromatograms). Figure 10 , Figure 11 ).

[0124] Table 4 shows the peak elution time and resolution results for each peak.

[0125] Peak Name Peak time / min Resolution GlcNAc 2.342 6.12 lactose 3.050 1.63 LNTⅡ 3.215 1.75 UDP-GlcNAc 3.598 8.13 GlcNAc-1-P 6.192 3.54 UMP 7.887 1.71 AMP 8.920 7.66 UDP 15.585 1.84 ADP 17.682 4.31 UTP 34.423 1.62 ATP 27.002 /

[0126] ②Sensitivity test

[0127] Using a stepwise dilution method and the detection method provided in Example 3, solutions of different concentrations of GlcNAc, lactose, GlcNAc-1-P, UDP-GlcNAc, ATP, AMP, ADP, UTP, UDP, UMP, and LNTII were analyzed. The lowest quantifiable concentrations of GlcNAc, lactose, GlcNAc-1-P, UDP-GlcNAc, ATP, AMP, ADP, UTP, UDP, UMP, and LNTII were determined under a signal-to-noise ratio of not less than 10. Specific results are shown in Table 5 (graphs are shown in Table 5). Figures 23-28 The results show that the detection method of the present invention has high sensitivity.

[0128] Table 5. Sensitivity test results

[0129] Substance Name Limit of Quantification Concentration Signal-to-noise ratio GlcNAc 0.002 mg / mL 24.8 lactose 0.008 mg / mL 19.3 GlcNAc-1-P 0.0025 mg / mL 18.0 UDP-GlcNAc 0.001 mg / mL 25.3 ATP 0.00125 mg / mL 12.1 AMP 0.001 mg / mL 30.0 ADP 0.001 mg / mL 13.0 UTP 0.0025 mg / mL 13.1 UDP 0.00125 mg / mL 12.1 UMP 0.001 mg / mL 17.7 LNTⅡ 0.008 mg / mL 23.6

[0130] ③ Linear examination

[0131] GlcNAc linear solution: Accurately weigh 20 mg of GlcNAc reference standard, place it in a 10 mL volumetric flask, add 70 vol% acetonitrile aqueous solution to dissolve and dilute to the mark, shake well; dilute stepwise to prepare a linear solution with GlcNAc concentration in the range of 0.002 to 2.0 mg / mL.

[0132] Linear lactose solution: Accurately weigh 20 mg of lactose reference standard, place it in a 10 mL volumetric flask, add 70 vol% acetonitrile aqueous solution to dissolve and dilute to the mark, shake well; dilute stepwise to prepare a linear solution with a lactose concentration in the range of 0.05–2.0 mg / mL.

[0133] GlcNAc-1-P linear solution: Accurately weigh 20 mg of GlcNAc-1-P reference standard, place it in a 10 mL volumetric flask, add 70 vol% acetonitrile aqueous solution to dissolve and dilute to the mark, shake well; dilute stepwise to prepare a linear solution with a GlcNAc-1-P concentration in the range of 0.0025 to 2.0 mg / mL.

[0134] UDP-GlcNAc linear solution: Accurately weigh 20 mg of UDP-GlcNAc reference standard, place it in a 10 mL volumetric flask, add 70 vol% acetonitrile aqueous solution to dissolve and dilute to the mark, shake well; dilute stepwise to prepare a linear solution with UDP-GlcNAc concentration in the range of 0.001 to 2.0 mg / mL.

[0135] ATP linear solution: Accurately weigh 20 mg of ATP, place it in a 10 mL volumetric flask, add 70 vol% acetonitrile aqueous solution to dissolve and dilute to the mark, shake well; dilute stepwise to prepare a linear solution with an ATP concentration in the range of 0.00125–2.0 mg / mL.

[0136] AMP linear solution: Accurately weigh 20 mg of AMP reference standard, place it in a 10 mL volumetric flask, add 70 vol% acetonitrile aqueous solution to dissolve and dilute to the mark, shake well; dilute stepwise to prepare a linear solution with AMP concentration in the range of 0.001 to 2.0 mg / mL.

[0137] ADP linear solution: Accurately weigh 20 mg of ADP reference standard, place it in a 10 mL volumetric flask, add 70 vol% acetonitrile aqueous solution to dissolve and dilute to the mark, shake well; dilute stepwise to prepare a linear solution with an ADP concentration in the range of 0.001 to 2.0 mg / mL.

[0138] UTP linear solution: Accurately weigh 20 mg of UTP reference standard, place it in a 10 mL volumetric flask, add 70 vol% acetonitrile aqueous solution to dissolve and dilute to the mark, shake well; dilute stepwise to prepare a linear solution with UTP concentration in the range of 0.0025 to 2.0 mg / mL.

[0139] UDP linear solution: Accurately weigh 20 mg of UDP reference standard, place it in a 10 mL volumetric flask, add 70 vol% acetonitrile aqueous solution to dissolve and dilute to the mark, shake well; dilute stepwise to prepare a linear solution with UDP concentration in the range of 0.0025 to 2.0 mg / mL.

[0140] UMP linear solution: Accurately weigh 20 mg of UMP reference standard, place it in a 10 mL volumetric flask, add 70 vol% acetonitrile aqueous solution to dissolve and dilute to the mark, shake well; dilute stepwise to prepare a linear solution with UMP concentration in the range of 0.001 to 2.0 mg / mL.

[0141] LNTⅡ linear solution: Accurately weigh 20 mg of LNTⅡ reference standard, place it in a 10 mL volumetric flask, add 70 vol% acetonitrile aqueous solution to dissolve and dilute to the mark, shake well; dilute stepwise to prepare a linear solution with LNTⅡ concentration in the range of 0.0125 to 2.0 mg / mL.

[0142] The analysis was performed using the detection method provided in Example 3, and a standard curve was plotted with concentration on the x-axis and peak area on the y-axis (see Example 3). Figures 12-22 The standard curve for GlcNAc is y = 33.856x - 0.4323, and the correlation coefficient Rc is [value missing]. 2 =0.9999; the standard curve for lactose is y = 1.0009x + 0.0063, with a correlation coefficient R. 2 =1.0000; the standard curve for GlcNAc-1-P is y = 25.031x + 0.126, and the correlation coefficient R0 is 1.0000. 2 =0.9999; the standard curve for UDP-GlcNAc is y = 91.9210x + 0.8978, and the correlation coefficient R0 is 0.9999. 2 =0.9999; the standard curve for ATP is y = 210.2x - 0.1875, and the correlation coefficient R is 0.9999. 2 =1.0000; the standard curve for ADP is y = 213.68x + 0.7036, and the correlation coefficient R0 is 1.0000. 2 =1.0000; the standard curve for AMP is y = 274.13x + 0.1162, and the correlation coefficient R0 is 1.0000. 2 =0.9999; the standard curve for UTP is y = 87.454x + 0.4282, and the correlation coefficient R0 is 0.9999. 2=0.9997; the standard curve for UDP is y = 115.8x + 0.9342, and the correlation coefficient R0 is 0.9997. 2 =0.9998; the standard curve for UMP is y = 135.13x + 0.2905, and the correlation coefficient R0 is 0.9998. 2 =0.9999; the standard curve for LNTⅡ is y = 10.032x + 0.0962, and the correlation coefficient R is 0.9999. 2 =0.9996; therefore, it can be seen that the correlation coefficient of each standard curve is not less than 0.999, and the standard curves all have a good linear relationship.

[0143] ④ System Applicability Assessment

[0144] Accurately weigh 25 mg each of GlcNAc, lactose, GlcNAc-1-P, UDP-GlcNAc, ATP, AMP, ADP, UTP, UDP, UMP, and LNTII reference standards, place them in a 50 mL volumetric flask, dissolve and dilute to the mark with 70% acetonitrile aqueous solution, shake well, and filter through a 0.22 μm organic phase filter membrane. Inject the sample six times consecutively using the detection method provided in Example 3, and calculate the peak area RSD value, which should be ≤2.0%. Specific results are shown in Table 6.

[0145] Table 6. Results of System Applicability Assessment

[0146]

[0147] Therefore, it can be seen that the method of the present invention has good system adaptability, and the RSD of the peak area of ​​each substance after repeated injection is less than 2.0%, which meets the requirements of quantitative accuracy.

[0148] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for the intermediate-control detection of enzyme-catalyzed synthesis of LNT II, ​​characterized in that, The process includes the following: During the enzyme-catalyzed synthesis of intermediates and / or final products of LNT II, ​​the reaction solution after inactivation and protein removal was detected by HPLC-UV-RI, and the detection results were quantitatively analyzed. The chromatographic conditions for HPLC-UV-RI detection are as follows: the chromatographic column is a polymer-based HILIC column, the column temperature is 50~60℃, mobile phase A is acetonitrile, mobile phase B is a dipotassium hydrogen phosphate aqueous solution with a concentration of 40~60 mmol / L, a pH value of 6.5~7.5, and a volume ratio of mobile phase A to mobile phase B of (65~75):(35~25). During the HPLC-UV-RI detection process, the substances measured include acetylglucosamine, lactose, acetylglucosamine-1-phosphate, adenosine triphosphate, adenosine monophosphate, adenosine diphosphate, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine 5'-bisphosphate-N-acetylglucosamine, and lactose-N-trisaccharide II.

2. The central control detection method according to claim 1, characterized in that, The specific synthetic route for the enzyme-catalyzed synthesis of LNT II is as follows: S1) Using acetylglucosamine and adenosine triphosphate as substrates, the first intermediate is generated by acetylglucosamine kinase catalysis. The first intermediate is acetylglucosamine-1-phosphate. S2) Using the first intermediate and uridine triphosphate as substrates, a second intermediate is generated by catalysis of acetylglucosamine-1-P uridine transferase. The second intermediate is uridine 5'-bisphosphate-N-acetylglucosamine. S3) Using the second intermediate and lactose as substrates, the final product lactose-N-trisaccharide II is synthesized by catalysis of acetylglucosamine transferase.

3. The central control detection method according to claim 1, characterized in that, The column temperature is 55°C.

4. The central control detection method according to claim 1, characterized in that, The concentration of the dipotassium hydrogen phosphate aqueous solution is 50 mmol / L, and the pH value of the dipotassium hydrogen phosphate aqueous solution is 7.

5. The central control detection method according to claim 1, characterized in that, The volume ratio of mobile phase A to mobile phase B is 70:

30.

6. The central control detection method according to claim 1, characterized in that, The specific method for inactivating and removing proteins is as follows: the reaction solution is mixed with an acetonitrile aqueous solution, followed by filtration.

7. The central control detection method according to claim 6, characterized in that, The concentration of the acetonitrile aqueous solution is 65~75 vol%; the pore size of the filter membrane is 0.15~0.3 μm.

8. The central control detection method according to claim 1, characterized in that, During the HPLC-UV-RI detection process, the wavelength of the ultraviolet detector used is 205~215nm.

9. The central control detection method according to claim 1, characterized in that, During the HPLC-UV-RI detection process, the temperature of the differential refractive index detector used is 35~45℃.

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