Covalent complex of human milk oligosaccharide and fluorescent reagent as well as preparation and application of covalent complex

Abstract

The invention provides a human milk oligosaccharide-fluorescence labeled conjugate and a preparation method thereof, and belongs to the field of organic chemistry. The human milk oligosaccharide-anthocyanin Cy7 conjugate provided by the invention is applied to tissue and organ fluorescence imaging, has a good tracing effect, and especially for dynamically tracing the in-vivo metabolic process of human milk oligosaccharide at different time points, compared with a traditional liquid chromatography-mass spectrometry method, the tracing method is accurate, visual and efficient, and the tracing effect is good. The method has potential advantages for judging whether the human milk oligosaccharide enters an intestinal barrier, a blood brain barrier and an in-vivo enriched part or not.

Description

Technical Field

[0001] The present invention relates to a covalent complex of human milk oligosaccharide and a fluorescent reagent, and the preparation and application thereof. Background Art

[0002] Human milk oligosaccharides (HMOs) are important components of breast milk and have important uses in promoting intestinal health, enhancing immune function, and aiding brain development. They can be used as ingredients that simulate breast milk in infant formula, functional foods and health products, and as carriers in the pharmaceutical field.

[0003] Typically, fluorescent labeling and tracing can clarify the metabolic pathways and mechanisms of action of different types of human milk oligosaccharides in infants. This can guide the optimization of infant formula design, aligning it more closely with the composition and function of breast milk, improving infant digestion and absorption, and reducing the incidence of allergies, diarrhea, and other problems. Fluorescence imaging can also be used to observe how human milk oligosaccharides are utilized by the intestinal microbiota and their impact on microbial structure and function. This can facilitate the development of human milk oligosaccharide-based intestinal microecological modulators to prevent and treat infant diseases associated with intestinal dysbiosis, such as antibiotic-associated diarrhea. By tracking their metabolism in vivo, we can gain insights into their specific mechanisms of immune regulation, providing a scientific basis for the development of functional foods or health supplements to enhance infant immunity. Fluorescence labeling and imaging can clearly demonstrate the interactions between human milk oligosaccharides and pathogens, providing clues for the development of new anti-infective drugs or therapeutic strategies, with particular potential for preventing and treating intestinal infections in infants and young children. Fluorescent labeling technology can be used to monitor the stability and changes of human milk oligosaccharides during food processing and storage, ensuring the content and activity of the active ingredients in the product and providing a scientific means for food quality control. Fluorescently labeled human milk oligosaccharides provide a powerful tool for studying basic biological processes such as intestinal physiology, metabolism, and immunity. By observing their metabolism and distribution in the body, we can gain a deeper understanding of the interactions between intestinal cells and microorganisms, as well as the mechanisms by which the intestinal microecology affects overall health. Metabolic research on human milk oligosaccharides can provide new targets and ideas for drug development. For example, developing drugs that can simulate or enhance the functions of human milk oligosaccharides, or using human milk oligosaccharides as drug carriers to improve the targeting and efficacy of drugs.

[0004] Based on the above, a general method was designed to design a series of fluorescently labeled human milk oligosaccharides from lactose to trace its metabolic process in the body, and its function was evaluated by fluorescence imaging, which has many important practical significances for deeply expanding the practical application of human milk oligosaccharides. Summary of the Invention

[0005] The purpose of the present invention is to provide a human milk oligosaccharide-fluorescent label covalent complex and a preparation method thereof;

[0006] At the same time, the present invention provides a method for tracing the metabolism of human milk oligosaccharides in vivo.

[0007] Technical Solution

[0008] Based on the purpose of the present invention, the first aspect of the present invention provides a covalent complex of human milk oligosaccharides and fluorescent markers, wherein the fluorescent reagent is a cyanine dye;

[0009] According to any embodiment of the first aspect of the present invention, the fluorescent reagent is anthocyanin Cy7 (Cyanine 7);

[0010] According to any embodiment of the first aspect of the present invention, the human milk oligosaccharides are lactose (Lac) with a mass-to-charge ratio of 342.11621 and connected with galactose and glucose; and 3' sialyllactose (3'Sialyllactose, 3'SL) and 6' sialyllactose (6'Sialyllactose, 6'SL) synthesized enzymatically based on lactose, with a mass-to-charge ratio of 632.20435 and sialic acid connected to lactose with α2-3 or α2-6 bonds.

[0011] According to any embodiment of the first aspect of the present invention, the covalent complex of human milk oligosaccharide and fluorescent marker has a structure represented by formula (I), (II) or (III):

[0012]

[0013]

[0014] As described herein, fluorescent reagents actually include, for example, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, etc. In the present invention, it was found that the use of anthocyanin Cy7 (Cyanine 7) as a fluorescent reagent can avoid the possibility that the coat color of C57BL / 6 mice (with black coat color) in other bands may have spontaneous fluorescence that affects the active imaging effect of the fluorescent probe. The Cy7 dye emits fluorescence in the near-infrared band, and the coat color of mice in this band does not excite fluorescence.

[0015] The second aspect of the present invention provides a method for preparing a covalent complex of human milk oligosaccharides and a fluorescent marker, comprising the steps of:

[0016] A solution containing human milk oligosaccharide-azide, anthocyanin Cy7, CuSO4, tris(3-hydroxypropyltriazolylmethyl)amine (hereinafter abbreviated as THPTA), and vitamin C is prepared, stirred, dried, and purified to obtain a covalent complex of human milk oligosaccharide and a fluorescent marker (i.e., human milk oligosaccharide-anthocyanin Cy7 conjugate);

[0017] The molar ratio of human milk oligosaccharide-azide, anthocyanin Cy7, CuSO4, THPTA and vitamin C is 1:(1.2-1.5):(1.2-1.5):(0.5-1):(2.5-3).

[0018] According to any embodiment of the second aspect of the present invention, the product comprises:

[0019] Any one or two of 3'-sialyllactose-anthocyanidin Cy7 conjugate, 6'-sialyllactose-anthocyanidin Cy7 conjugate, and lactose-anthocyanidin Cy7 conjugate.

[0020] According to any embodiment of the second aspect of the present invention, the stirring is carried out at room temperature in the dark for 4-6 hours.

[0021] According to any embodiment of the second aspect of the present invention, the human milk oligosaccharide-azide comprises:

[0022] 3'-sialyllactose-azide; and / or,

[0023] 6'-sialyllactose-azide; and / or,

[0024] Lactose-azide.

[0025] According to any embodiment of the second aspect of the present invention, the stirring is carried out at room temperature in the dark for 4-6 hours.

[0026] According to any embodiment of the second aspect of the present invention, the drying is freeze-drying (lyophilization).

[0027] According to any embodiment of the second aspect of the present invention, the purification comprises: resuspending the freeze-dried (lyophilized) product, and then purifying it on a silica gel column or a polyacrylamide gel column (Bio-Gel P-2).

[0028] According to any embodiment of the second aspect of the present invention, the method for preparing the covalent complex of human milk oligosaccharide and fluorescent marker comprises the following detailed steps:

[0029] Dissolve 3'-sialyllactose-azide and / or 6'-sialyllactose-azide and / or lactose-azide, anthocyanidin Cy7, CuSO4, THPTA, and vitamin C in water at a molar ratio of 1:(1.2-1.5):(1.2-1.5):(0.5-1):(2.5-3);

[0030] Stir at room temperature in the dark for 4-6 hours;

[0031] After freeze-drying, the mixture is resuspended, purified on a silica gel column or a polyacrylamide gel column (Bio-Gel P-2), and freeze-dried to obtain a covalent complex of human milk oligosaccharides and a fluorescent marker (i.e., human milk oligosaccharides-anthocyanidin Cy7 conjugate).

[0032] According to any embodiment of the second aspect of the present invention, the 3'-sialyllactose-azide / 6'-sialyllactose-azide is prepared by the following steps:

[0033] A solution containing lactose-azide, sialic acid, MgCl2, Tris-HCl, and cytidine triphosphate (CTP) is prepared and mixed with CMP-sialic acid synthase (NmCSS) and sialyltransferase.

[0034] Incubate at constant temperature to obtain 3'-sialyllactose-azide / 6'-sialyllactose-azide.

[0035] According to any embodiment of the second aspect of the present invention, the molar ratio of lactose-azide, sialic acid, MgCl2, Tris-HCl, and CTP is 1:(1.2-1.5):(1-2):(5-7):(1.2-1.5).

[0036] According to any embodiment of the second aspect of the present invention, the constant temperature incubation time is 3 to 6 hours; the constant temperature incubation temperature is 34 to 38°C.

[0037] According to any embodiment of the second aspect of the present invention, the solvent is water.

[0038] According to any embodiment of the second aspect of the present invention, the amount of the protease NmCSS is 3-6 uM.

[0039] According to any embodiment of the second aspect of the present invention, the amount of the sialyltransferase used is 1-2 uM.

[0040] According to any embodiment of the second aspect of the present invention, the sialyltransferase is α2-3 sialyltransferase (abbreviated as tpd2,3ST) and α2-6 sialyltransferase (abbreviated as tpd2,6ST).

[0041] According to any embodiment of the second aspect of the present invention, the method for preparing the covalent complex of human milk oligosaccharide and fluorescent marker comprises the following detailed steps:

[0042] (1) Lactose-azide, sialic acid, MgCl2, Tris-HCl (8.5), and CTP are dissolved in a solvent at a molar ratio of 1:(1.2-1.5):(1-2):(5-7):(1.2-1.5), protease NmCSS and tpd2,3ST / tpd2,6ST are added, and the mixture is incubated at a constant temperature to obtain a reaction product, wherein the reaction product contains 3'-sialyllactose-azide and / or 6'-sialyllactose-azide and / or human milk oligosaccharide-azide;

[0043] The reaction product, 3'-sialyllactose-azide or 6'-sialyllactose-azide, was purified on a silica gel column using a mixture of ethyl acetate, methanol, water, and acetic acid as the developing solvent, with the most preferred ratio being ethyl acetate:methanol:water:acetic acid = 4:2:1:0.2. The product was determined by TLC at the time of product elution, and the product was collected, rotary evaporated, and lyophilized to obtain the product.

[0044] (2) The product obtained in step 1 (3'-sialyllactose-azide, 6'-sialyllactose-azide or lactose-azide) is dissolved in water with anthocyanin Cy7, CuSO4, THPTA and vitamin C at a molar ratio of (1.2-1.5):(1.2-1.5):(0.5-1):(2.5-3), and stirred in the dark at room temperature for 4-6 hours; after freeze-drying, the mixture is resuspended, purified on a silica gel column or a polyacrylamide gel column (Bio-GelP-2), and freeze-dried to obtain the reaction product, human milk oligosaccharide-anthocyanin Cy7 conjugate.

[0045] A third aspect of the present invention provides a method for tracing the metabolism of human milk oligosaccharides in vivo, using a covalent complex of human milk oligosaccharides and a fluorescent marker provided by any embodiment of the first aspect of the invention to trace the metabolism of human milk oligosaccharides in vivo; or, using a covalent complex of human milk oligosaccharides and a fluorescent marker prepared by the method provided by any embodiment of the second aspect of the invention to trace the metabolism of human milk oligosaccharides in vivo.

[0046] Any embodiment of any aspect of the present invention can be combined with other embodiments without contradiction. In addition, in any embodiment of any aspect of the present invention, any technical feature can be applied to the technical feature in other embodiments without contradiction.

[0047] Provided that no contradiction arises, any technical feature of any aspect of the present invention or any embodiment of such aspect is also applicable to any other embodiment or any embodiment of any other aspect. Of course, when applicable to each other, the corresponding features may be appropriately modified as necessary. The various aspects and features of the present invention are further described below.

[0048] 3. Beneficial effects

[0049] Cy7 is a widely used in vivo fluorescent labeling reagent in animal experiments. It can detect its functional activity by connecting it to the bioactive molecule to be tested through orthogonal chemical methods. It has important advantages such as high detection sensitivity, intuitiveness, and the ability to dynamically track metabolic processes by in vivo imaging. The present invention aims to design a universal method to design a series of fluorescently labeled human milk oligosaccharides using lactose to track its metabolic process in vivo. Through the efficient method of fluorescence imaging, the functions of various types of human milk oligosaccharides are evaluated, and the practical application of human milk oligosaccharides is deeply expanded; it can solve the problem of tracing the metabolism of human milk oligosaccharides in vivo. More specifically:

[0050] (1) The human milk oligosaccharide-fluorescent labeling conjugate provided by the present invention and its preparation method. The human milk oligosaccharide-anthocyanidin Cy7 conjugate provided by the present invention can be used in tissue and organ fluorescence imaging;

[0051] (2) The human milk oligosaccharide-fluorescently labeled conjugate human milk oligosaccharide provided by the present invention has a good tracing effect, especially for dynamically tracing the metabolic process of human milk oligosaccharides in the body at different time points. Compared with the traditional liquid chromatography-mass spectrometry method, this tracing method is accurate, intuitive, and efficient, and has potential advantages in determining whether human milk oligosaccharides have entered the intestinal barrier, the blood-brain barrier, and the enriched part in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Spectrum of the crude product before 6'SL-Cy7 purification;

[0053] Figure 2 Mass spectrum of lactose-anthocyanidin Cy7;

[0054] Figure 3 Mass spectrum of the purified 6'SL-Cy7 standard;

[0055] Figure 4 Data graph of Cy7, Lac-Cy7, and 6'SL-Cy7 for in vivo imaging of small animals;

[0056] Figure 5 Data graph of Cy7, Lac-Cy7, and 6'SL-Cy7 for in vivo imaging of small animals;

[0057] Figure 6 Histogram of fluorescence intensity on the back and abdomen of mice;

[0058] Figure 7 Fluorescence signal imaging of Cy7, Lac-Cy7, and 6'SL-Cy7 in the small and large intestines;

[0059] Figure 8 Fluorescence signal imaging of Cy7, Lac-Cy7, and 6'SL-Cy7 in various organs;

[0060] Figure 9 Statistical diagram of the retention rates of Cy7, Lac-Cy7, and 6'SL-Cy7 in the small intestine and large intestine;

[0061] Figure 10 Fluorescence signal imaging and retention rate statistics of Cy7, Lac-Cy7, and 6'SL-Cy7 in the brain;

[0062] Figure 11 6'SL-Cy7 was detected in mouse brain tissue after oral administration;

[0063] Figure 12 Dynamic changes of 6'SL-Cy fluorescence intensity over time in the small intestine and large intestine;

[0064] Figure 13 Fluorescence signal imaging of 6'SL-Cy7 in various organs;

[0065] Figure 14 Statistical diagram of the retention rate of 6'SL-Cy7 in various organs;

[0066] Figure 15 Fluorescence signal imaging of 6'SL-Cy7 in various organs;

[0067] Figure 16 Distribution of 6'SL-Cy7 in different organs;

[0068] Figure 17 Dynamic distribution of 6'SL-Cy7 fluorescence intensity over time in the whole, top, and bottom regions of the mouse brain;

[0069] Figure 18 Figure a shows the distribution of fluorescence signals in various organs of mice 0.5 hours after oral gavage of normal saline, Lac-Cy7, and 6'SL-Cy7; Figure b shows the distribution of fluorescence signals in the small intestine and large intestine of mice 0.5 hours after oral gavage of normal saline, Lac-Cy7, and 6'SL-Cy7;

[0070] Figure 19 Distribution of fluorescent signals in the brain of mice 0.5 h after oral gavage with saline, Lac-Cy7, and 6'SL-Cy7. DETAILED DESCRIPTION

[0071] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0072] All documents cited herein are incorporated herein by reference in their entirety, and if the meanings expressed in these documents are inconsistent with those of the present invention, the present invention shall prevail. In addition, various terms and phrases used in the present invention have the general meanings known to those skilled in the art. Even so, the present invention still intends to provide a more detailed description and explanation of these terms and phrases herein. If the terms and phrases mentioned are inconsistent with the generally known meanings, the meanings expressed in the present invention shall prevail.

[0073] In the present invention, the terms "comprising" or "containing" or "including" or "include" indicate that various components can be used together in the composition of the present invention. Therefore, the terms "consisting mainly of..." and "consisting of..." are included in the terms "comprising" or "containing".

[0074] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0075] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement, or value. One skilled in the art can readily determine the degree of flexibility for a particular variable.

[0076] Concentration, content, percentage composition and other numerical data can be presented in range format in this article.Should be understood that such range format is only used for convenience and brevity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all independent numerical values ​​or subranges encompassed within the scope, just as each numerical value and subrange are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including independent numerals (such as 2,3,4) and subranges (such as 1 to 3,2 to 4 etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all above-mentioned values ​​and scopes.In addition, no matter how the breadth of described scope or feature is, this explanation should be applicable.

[0077] The present invention will be further described below with reference to specific examples. In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturer.

[0078] Data statistical analysis

[0079] All experimental data are expressed as mean ± standard error. Data were analyzed using Graph Pad Prism 7.0, using one-way analysis of variance and Bonferroni post hoc statistical tests for multiple comparisons or two-sided tests. P < 0.05 was considered to indicate a significant difference between groups.

[0080] Statistical analysis

[0081] Data were analyzed by Prism 7.0 (GraphPad Software Inc., San Diego, CA) using one-way analysis of variance (ANOVA) with Bonferroni's post-test for comparison of more than two data sets, with statistical significance accepted at p < 0.05.

[0082] Reagents

[0083] 6'-sialyllactose, purity: 99%%;

[0084] 3'-sialyllactose, purity: 99%%;

[0085] Anthocyanin Cy7 was purchased from Shaanxi Xinyan Bomei Biotechnology Co., Ltd., brand / product number: Xinyan Bomei / X-CL-1391;

[0086] C57BL / 6 mice (20 g) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., China. The pups were fed by their mothers until weaning on the 19th day after birth, and experiments were started on the 21st day. Other reagents can be purchased commercially.

[0087] It is further explained that the human milk oligosaccharide-anthocyanidin Cy7 conjugate provided by the present invention includes sialyllactose-anthocyanidin Cy7 and lactose-anthocyanidin Cy7, wherein sialyllactose-anthocyanidin Cy7 at least includes 3'-sialyllactose-anthocyanidin Cy7 chemical conjugate and 6'-sialyllactose-anthocyanidin Cy7 chemical conjugate.

[0088] Wherein, the lactose-anthocyanidin Cy7 (abbreviated as Lac-Cy7) is represented by the following formula (I):

[0089]

[0090] The preparation steps of Lac-Cy7 for reference are as follows:

[0091] Lactose-azide is dissolved in water with anthocyanin Cy7, CuSO4, THPTA, and vitamin C at a molar ratio of 1:(1.2-1.5):(1.2-1.5):(0.5-1):(2.5-3), and stirred in the dark at room temperature for 4-6 hours; after freeze-drying, the mixture is resuspended, purified on a silica gel column or a polyacrylamide gel column (Bio-Gel P-2), and freeze-dried to obtain the reaction product, 6'-sialyllactose-anthocyanin Cy7 conjugate.

[0092] The reaction product is green, and the reaction rate of the product can basically reach 98%.

[0093] The 3'-sialyllactose-anthocyanidin Cy7 (abbreviated as 3'SL-Cy7) is shown in the following formula (II):

[0094]

[0095] The preparation steps for 3'SL-Cy7 are as follows:

[0096] The preparation steps for 3'SL-Cy7 are as follows:

[0097] (1) Lactose-azide, sialic acid, MgCl2, Tris-HCl (8.5), and CTP are dissolved in water at a molar ratio of 1: (1.2-1.5): (1-2): (5-7): (1.2-1.5), 3-6 μM protease NmCSS and 1-2 μM tpd2,3ST are added, and the mixture is reacted at 37° C. for 8-10 h to obtain a reaction product, which is 3'-sialyllactose-azide;

[0098] The reaction product, 3'-sialyllactose-azide, was purified on a silica gel column using a developing solvent consisting of ethyl acetate:methanol:water:acetic acid (4:2:1:0.2). The product was determined by TLC at the time of product eluent release. The product was then collected by rotary evaporation and lyophilized to obtain the product.

[0099] (2) The product obtained in step 1 (3'-sialyllactose-azide) is dissolved in water with anthocyanin Cy7, CuSO4, THPTA, and vitamin C at a molar ratio of 1:(1.2-1.5):(1.2-1.5):(0.5-1):(2.5-3), and stirred in the dark at room temperature for 4-6 hours; after freeze-drying, the mixture is resuspended, purified on a silica gel column or a polyacrylamide gel column (Bio-Gel P-2), and freeze-dried to obtain the reaction product 3'SL-Cy7.

[0100] 6'-sialyllactose-anthocyanidin Cy7 (abbreviated as 6'SL-Cy7) is shown in the following formula (III):

[0101]

[0102] The preparation steps for 6'SL-Cy7 are as follows:

[0103] (1) Lactose-azide, sialic acid, MgCl2, Tris-HCl (8.5), and CTP are dissolved in water at a molar ratio of 1: (1.2-1.5): (1-2): (5-7): (1.2-1.5), 3-6 μM protease NmCSS and 1-2 μM tpd2,6ST are added, and the mixture is reacted at 37° C. for 8-10 h to obtain a reaction product, which is 6'-sialyllactose-azide;

[0104] The reaction product, 6'-sialyllactose-azide, was purified on a silica gel column using a developing solvent consisting of ethyl acetate:methanol:water:acetic acid (4:2:1:0.2). The product was determined by TLC at the time of product eluent release. The product was then collected by rotary evaporation and lyophilized to obtain the product.

[0105] (2) The product obtained in step 1 (6'-sialyllactose-azide) is dissolved in water with anthocyanin Cy7, CuSO4, THPTA, and vitamin C at a molar ratio of 1:(1.2-1.5):(1.2-1.5):(0.5-1):(2.5-3), and stirred in the dark at room temperature for 4-6 hours; after freeze-drying, the mixture is resuspended, purified on a silica gel column or a polyacrylamide gel column (Bio-Gel P-2), and freeze-dried to obtain the reaction product 6'SL-Cy7.

[0106] The reaction product is green, and the reaction rate of the product can basically reach 98%.

[0107] Among them, the polyacrylamide gel column (Bio-Gel P-2) purification is a conventional purification method in the field. It is a process of separation in the mobile phase according to molecular weight. It can be used to separate solutes of different molecular weights. Substances with lower molecular weight have a long retention time, while substances with higher molecular weight are discharged earlier. The polyacrylamide gel is a conventional high-resolution gel filtration in the field. The molecular sieve purification range is 100-100kDa. The polyacrylamide gel column (Bio-Gel P-2) is used to remove unreacted anthocyanin Cy7 and small molecule salts. The purification time is 3 to 4 hours, and the reaction product is visible in green.

[0108] The drying method is conventional in the art. Preferably, the drying method is vacuum freeze drying, vacuum drying, spray drying, oven drying, or infrared drying, with vacuum freeze drying being most preferred. The vacuum freeze drying parameters are: temperature of -50 to -80°C, vacuum degree of 20 to 30 Pa, time of 36 to 48 hours, and protection from light.

[0109] Example 1

[0110] Preparation of 6'-sialyllactose-anthocyanidin Cy7 conjugate

[0111] The reaction equation of 6'-sialyllactose-anthocyanidin Cy7 conjugate is shown in Scheme 1-1 below:

[0112] Compound 1 is lactose-azide (Lac-N3 in Scheme 1-1), which reacts with compound 2, sialic acid (Sia in Scheme 1-1), to obtain 6'-sialyllactose-azide, namely compound 3 (6'SL-N3 in Scheme 1-1);

[0113] The azide on compound 3 reacts with the alkyne of compound 4 (anthocyanidin, Cy7 in Scheme 1-1) to form a ring to obtain compound 5 (6'SL-Cy7 in Scheme 1-1), which is the 6'-sialyllactose-anthocyanidin Cy7 conjugate.

[0114]

[0115] The specific implementation steps of the chemical coupling reaction in this embodiment are as follows:

[0116] (1) Lactose-azide, sialic acid, MgCl2, Tris-HCl (8.5), and CTP were dissolved in water at a molar ratio of 1:1.22:2:5.56:1.22. The protease NmCSS (concentration 3 μM) and tpd2,6ST (concentration 1 μM) were added and reacted at 37°C for 8 h. The reaction product, 6'-sialyllactose-azide (6'SL-N3), was purified on a silica gel column using a developing solvent of ethyl acetate:methanol:water:acetic acid = 4:2:1:0.2. The product elution time was determined by TLC. The product was collected and evaporated under reduced pressure at 30°C, resuspended in water, and lyophilized.

[0117] (2) The product 6'-sialyllactose-azide obtained in step 1 is dissolved in water with anthocyanin Cy7, CuSO4, THPTA, and vitamin C at a molar ratio of 1:1.2:0.4:2:2, and stirred at room temperature in the dark for 4 hours; after freeze-drying, the mixture is resuspended, purified on a polyacrylamide gel (Bio-Gel P-2) or a silica gel column, and freeze-dried to obtain the reaction product to prepare the 6'-sialyllactose-anthocyanin Cy7 conjugate.

[0118] The prepared 6'-sialyllactose-anthocyanidin Cy7 conjugate 6'SL-Cy7 was analyzed by mass spectrometry. The spectrum of the crude product of 6'SL-Cy7 before purification is as follows: Figure 1 As shown, the spectrum of the purified 6'SL-Cy7 standard is as follows Figure 3 As shown. Figure 1 、 3 The mass spectrum analysis results are as follows:

[0119] Figure 1 The 754.63 peak and Figure 3 The compound structure corresponding to the 754.27 peak is shown in the following formula (II), which corresponds to 6'-sialyllactose-anthocyanidin Cy7 conjugate 6'SL-Cy7:

[0120]

[0121] Figure 1 The compound structure corresponding to the peak at 791.30 is shown in the following formula (IV), which corresponds to 6'-sialyllactose-azide 6'SL-N3:

[0122]

[0123] Preparation of lactose-anthocyanidin Cy7 conjugate

[0124] The reaction equation of lactose-anthocyanidin Cy7 conjugate is shown in Scheme 1-2 below:

[0125] Compound 1 is lactose-azide (Lac-N3 in Scheme 1-2) which undergoes a cyclization reaction with the alkyne of compound 4 (anthocyanidin, Cy7 in Scheme 1-2) to obtain compound 6 (Lac-Cy7 in Scheme 1-2), which is a lactose-anthocyanidin Cy7 conjugate.

[0126]

[0127] The specific implementation steps of the chemical coupling reaction in this embodiment are as follows:

[0128] (1) Lactose-azide, anthocyanidin Cy7, CuSO4, THPTA, and vitamin C are dissolved in water at a molar ratio of 1:1.2:0.4:2:2, and stirred at room temperature in the dark for 4 hours; after freeze-drying, the mixture is resuspended, purified on a polyacrylamide gel (Bio-Gel P-2) or a silica gel column, and freeze-dried to obtain the reaction product to prepare the lactose-anthocyanidin Cy7 conjugate.

[0129] The prepared lactose-anthocyanidin Cy7 conjugate Lac-Cy7 was analyzed by mass spectrometry, and the spectrum was as follows Figure 2 As shown. Figure 2 The mass spectrum analysis results are as follows:

[0130] Figure 2 The compound structure corresponding to the peak at 608.23 is shown in the following formula (I), which corresponds to the lactose-anthocyanidin conjugate Lac-Cy7:

[0131]

[0132] Example 2

[0133] Human milk oligosaccharide-anthocyanin Cy7 conjugate for in vivo imaging of small animals

[0134] In this example, the in vivo imaging effect of the prepared human milk oligosaccharide - anthocyanidin Cy7 was studied in small animals. The C57BL / 6 mice used were from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., China. The young mice were fed by their mothers until weaning on the 19th day after birth. On the 21st day, the mice were gavaged with anthocyanidin Cy7 (abbreviated as Cy7), lactose-anthocyanidin Cy7 conjugate (Lac-Cy7), and 6'-sialyllactose-anthocyanidin Cy7 conjugate (abbreviated as 6'SL-Cy7). The mice were imaged in vivo before gavage, and at 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, and 12h after gavage.

[0135] See also Figure 4 and Figure 5 Figure 2 shows data from in vivo imaging of small animals using Cy7, Lac-Cy7, and 6'SL-Cy7. The experimental results demonstrate that the 6'SL-Cy7 group exhibits excellent tracing efficacy. Compared to the Cy7 group, 6'SL-Cy7 undergoes significantly faster metabolism, indicating that the fluorescent dye Cy7 does not affect the normal metabolism of 6'-sialyllactose.

[0136] like Figure 6 As shown, Figure 6 Figure C is a bar chart of fluorescence intensity on the back of mice; Figure 6 Figure D is a bar graph of fluorescence intensity in the mouse abdomen. The graph shows that 6'SL-Cy7 undergoes significant metabolism as early as 6 hours and is essentially cleared from the body by 12 hours compared to Cy7, indicating that 6'-sialyllactose does not accumulate in the body and has good biosafety.

[0137] like Figure 7 As shown, the fluorescence signal of 6'SL-Cy7 in the small and large intestines was weak at 12 hours, and it remained largely in the cecum. This suggests that after 12 hours, 6'-sialyllactose is largely excreted from the body, and the remaining portion may serve as a substrate for intestinal flora and continue to be used by intestinal flora.

[0138] like Figure 8 As shown, at 12 hours, Lac-Cy7 and 6'SL-Cy7 were primarily distributed in the liver and kidneys, with smaller amounts also in the spleen and lungs. This is because Lac-Cy7 and 6'SL-Cy7 enter the bloodstream via the portal vein, first accumulating in the liver, then being transported throughout the body and ultimately excreted through the kidneys, resulting in no toxicological effects.

[0139] like Figure 9As shown in the figure, the statistical results showed that the retention rates of Lac-Cy7 and 6'SL-Cy7 in the small intestine and large intestine were significantly lower than that of Cy7 at 12 hours, and the contents of Lac-Cy7 and 6'SL-Cy7 in the liver were also lower than that of Cy7, indicating that lactose and 6'-sialyllactose have good biosafety.

[0140] like Figure 10 As shown in the results, 6'SL-Cy7 was significantly enriched in the brain at 12 hours, and Lac-Cy7 was also relatively significantly enriched in the brain, indicating that 6'-sialyllactose is a potential donor of exogenous sialic acid during the infant brain development stage and plays an important role in promoting neural development.

[0141] See also Figure 11 , which is 6'SL-Cy7 detected in the mouse brain tissue after oral administration, indicating that the protein successfully entered the mouse brain.

[0142] Example 3 Fluorescence imaging effect of human milk oligosaccharide-anthocyanidin Cy7 conjugate on small animal tissues and organs.

[0143] In this example, the metabolic distribution of human milk oligosaccharide anthocyanin Cy7 in mouse tissues and organs at different time points was studied. C57BL / 6 mice were obtained from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., China. Pups were nursed by their mothers until weaning on day 19 after birth.

[0144] Cy7, Lac-Cy7, and 6'SL-Cy7 samples were prepared for this study. Weaned mice were gavage-administered 100 μL of a 300 μg / mL aqueous solution of each sample at day 21. The intestines and organs of the mice were dissected at 0.5, 1.5, 3, 6, and 12 hours post-mortem to evaluate the fluorescence signal imaging of 6'SL-Cy7.

[0145] See also Figure 12-16 , which shows the fluorescence imaging data of Cy7, Lac-Cy7 and 6'SL-Cy7 in the intestines and organs of mice at different time points. The experimental results show that at 0.5h, the fluorescence signal of 6'SL-Cy7 is mainly distributed in the upper digestive tract ( Figure 12 In addition, the fluorescence signal of 6'SL-Cy7 moved to the lower digestive tract over time, and the speed was faster and the peak value was higher in male mice than in female mice ( Figure 12 B and C).

[0146] like Figure 12 B-1 and C-1 represent the dynamic changes of 6'SL-Cy fluorescence intensity in the small intestine and large intestine over time, respectively. The blue broken line represents the 6'SL-Cy7 group, and the red broken line represents the control group.

[0147] Within 0.5 h of oral administration, all organs and the brain showed strong fluorescence signals, which even masked the signals at other time points ( Figure 13-15 Brain fluorescence reached its maximum value at 0.5h-1.5h and then dropped sharply. However, compared with the control group, the fluorescence signal could still be detected at 12h ( Figure 15 The fluorescence intensity of other organs gradually decreased over time, and at 12 hours, only the liver and kidneys still had a small amount of fluorescence, indicating that it had been basically cleared ( Figure 13-15 ).

[0148] like Figure 17 As shown, the dynamic distribution of the overall, top and bottom fluorescence intensity of 6'SL-Cy7 in the mouse brain over time. It can be seen from the figure that compared with the control group, 6'SL-Cy7 in the treated group mice significantly entered the brain.

[0149] like Figure 18 As shown, Figure a shows the distribution of fluorescence signals in various organs of mice 0.5 hours after oral gavage of normal saline, Lac-Cy7 and 6'SL-Cy7; Figure b shows the distribution of fluorescence signals in the small intestine and large intestine of mice 0.5 hours after oral gavage of normal saline, Lac-Cy7 and 6'SL-Cy7. Figure 19 As shown, the distribution of fluorescence signals in the brain of mice 0.5 hours after oral administration of normal saline, Lac-Cy7 and 6'SL-Cy7; Figure 18 and Figure 19 It can be seen that in the supplementary experiment of 6'SL entering the brain, the fluorescence signals of the Lac-Cy7 and 6'SL-Cy7 treatment groups were significantly enhanced compared with the control group, indicating that 6'SL-Cy7 can indeed pass through the blood-brain barrier.

[0150] The above experiments demonstrate efficient absorption of 6'SL by the mouse upper gastrointestinal tract. Orally administered 6'-sialyllactose enters the bloodstream via the portal vein and accumulates in the liver. It subsequently may cross the blood-brain barrier and accumulate in the brain, supplying the developing brain with essential sialic acid. Our results also showed that the metabolic rate of 6'SL-Cy7 was higher than that of the commonly used fluorescent dye Cy7, indicating that the body can efficiently break down and utilize 6'SL. This suggests a lower risk of accumulation over time, an important consideration for its use in therapeutic applications. Furthermore, male mice metabolized 6'SL faster than female mice. This intriguing finding suggests that sex-based differences in 6'SL metabolism may also exist in humans. Previous studies have demonstrated sex-based differences in drug metabolism, highlighting the potential implications of such differences for dosing and drug efficacy. In summary, 6'SL may have important physiological implications, including its potential to cross the blood-brain barrier and promote brain function.

[0151] The above description is merely an illustrative description of the present invention and its embodiments, which is not restrictive. The embodiment shown in the embodiment is only one embodiment of the present invention, and the actual embodiment is not limited thereto. Therefore, if a person skilled in the art is inspired by the above description and, without departing from the purpose of the present invention, designs an embodiment and examples similar to the technical solution without creatively designing, they shall fall within the scope of protection of the present invention.

Claims

1. A covalent complex of human milk oligosaccharides and a fluorescent marker, characterized in that: The fluorescent reagent is anthocyanin Cy7.

2. The covalent complex of human milk oligosaccharide and fluorescent marker according to claim 1, characterized in that Based on human lactose and sialyllactose; in, The mass-to-charge ratio of the lactose is 342.11621, and the lactose is connected by galactose and glucose; The mass-to-charge ratio of the sialyllactose is 632.20435, and the sialic acid is linked to the lactose via an α2-3 or α2-6 bond.

3. The covalent complex of human milk oligosaccharide and fluorescent marker according to claim 1, characterized in that The sialyllactose is 3'-sialyllactose and / or 6'-sialyllactose.

4. The covalent complex of human milk oligosaccharide and fluorescent marker according to any one of claims 1 to 3, characterized in that The covalent complex of human milk oligosaccharide and fluorescent marker has a structure shown in formula (I), (II) or (III):

5. The method for preparing a covalent complex of human milk oligosaccharide and a fluorescent marker according to any one of claims 1 to 4, characterized in that: Including steps: preparing a solution containing human milk oligosaccharide-azide and anthocyanin Cy7, CuSO4, tris(3-hydroxypropyltriazolylmethyl)amine, and vitamin C, stirring, drying, and purifying to obtain a covalent complex of human milk oligosaccharide and a fluorescent marker (i.e., human milk oligosaccharide-anthocyanin Cy7 conjugate); The molar ratio of human milk oligosaccharide-azide, anthocyanin Cy7, CuSO4, tris(3-hydroxypropyltriazolylmethyl)amine and vitamin C is 1:(1.2-1.5):(1.2-1.5):(0.5-1):(2.5-3).

6. The method for preparing a covalent complex of human milk oligosaccharides and fluorescent markers according to claim 5, characterized in that: The stirring is carried out at room temperature in the dark for 4-6 hours; and / or, The drying is freeze drying; and / or, Resuspending the freeze-dried (lyophilized) product and then purifying it on a silica gel column or a polyacrylamide gel column (Bio-Gel P-2); and / or, The human milk oligosaccharide-azide comprises: 3'-sialyllactose-azide; and / or, 6'-sialyllactose-azide; and / or, Lactose-azide.

7. The method for preparing a covalent complex of human milk oligosaccharides and fluorescent markers according to claim 6, characterized in that: The 3'-sialyllactose-azide / 6'-sialyllactose-azide is prepared by the following steps: A solution containing lactose-azide, sialic acid, MgCl2, Tris-HCl, and CTP is prepared and mixed with protease NmCSS and sialyltransferase; Incubate at constant temperature to obtain 3'-sialyllactose-azide / 6'-sialyllactose-azide. The molar ratio of lactose-azide, sialic acid, MgCl2, Tris-HCl and CTP is 1:(1.2-1.5):(1-2):(5-7):(1.2-1.5).

8. The method for preparing a covalent complex of human milk oligosaccharides and fluorescent markers according to claim 7, characterized in that: The constant temperature incubation time is 3 to 6 hours; and / or, The amount of the protease NmCSS used is 3-6 uM.

9. The method for preparing a covalent complex of human milk oligosaccharide and a fluorescent marker according to any one of claims 7 to 8, characterized in that: The dosage of the sialyltransferase is 1-2 uM.

10. A method for tracing the metabolism of human milk oligosaccharides in vivo, characterized in that: The metabolic tracing of human milk oligosaccharides in vivo is performed using the covalent complex of human milk oligosaccharides and fluorescent markers according to any one of claims 1 to 4, or the covalent complex of human milk oligosaccharides and fluorescent markers prepared by the method according to any one of claims 5 to 9.

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