CN-122011057-A - Partially acylated non-natural sugar for metabolic markers and application

Abstract

The invention discloses a partially acylated non-natural sugar for metabolic labeling and application thereof, wherein the partially acylated non-natural sugar is mannose type of pyranose structure, and 1-hydroxy and 6-hydroxy are protected by hydrophobic groups. The invention combines the advantages of the existing non-natural sugar, ensures that the non-natural sugar can be efficiently utilized by cells, effectively avoids S side reaction with cysteine in protein in the metabolic process of the non-natural sugar, and realizes efficient metabolic labeling. In the cell test, 1, 6-bisacylated unnatural saccharides were used at concentrations an order of magnitude lower than unnatural saccharides without protecting groups.

Inventors

• CHEN XING
• CHENG BO

Assignees

• 合肥领新博瑞生物科技有限公司

Dates

Publication Date

20260512

Application Date

20221027

Claims (10)

1. 1. A partially acylated unnatural saccharide for use in metabolic labelling, wherein the partially acylated unnatural saccharide is mannose-type of pyranose structure and the 1-hydroxy and 6-hydroxy groups are protected by hydrophobic groups.
2. 2. The partially acylated unnatural saccharide for metabolic labeling according to claim 1, wherein the hydrophobic group is any one of acetyl, propionyl, butyryl.
3. 3. The partially acylated non-natural saccharide for metabolic markers according to claim 1, wherein the partially acylated non-natural saccharide for metabolic markers is a 1, 6-bisacylated mannose non-natural saccharide having the structural formula: Wherein, R=CH 2 N 3 , n=0, 1 or 2, and the values of n at two positions are the same; or r=ch 2 CH 2 CCH, n=1; Or r=och 2 CCH, n=1.
4. 4. The partially acylated unnatural saccharide for a metabolic marker according to claim 3, wherein the 1, 6-bisacylated mannose type unnatural saccharide comprises 1,6-Ac 2 ManNAz、1,6-Pr 2 ManNAz、1,6-Bu 2 ManNAz、1,6-Pr 2 ManNAl and 1,6-Pr 2 ManNProc.
5. 5. The partially acylated unnatural saccharide for a metabolic marker according to claim 3, wherein the partially acylated unnatural saccharide for a metabolic marker is a1, 6-dipropionated mannose type unnatural saccharide.
6. 6. Use of a partially acylated non-native saccharide for metabolic labeling according to any one of claims 1-5 for saccharide metabolic labeling of any one of HeLa cells, 3T3 cells, CHO cells and MCF-7 cells.
7. 7. Use of the 1, 6-dipropionated mannose-type non-native saccharide of claim 5 for in vivo metabolic labelling of mice.
8. 8. A kit for the labeling of sugar metabolism comprising a partially acylated unnatural sugar for metabolic labeling according to any one of claims 1-5.
9. 9. The kit for labeling of sugar metabolism according to claim 8, wherein the kit is used for labeling of sugar metabolism of any one of HeLa cells, 3T3 cells, CHO cells and MCF-7 cells.
10. 10. A glycometabolism marker kit according to claim 8 or 9, wherein for glycometabolism marking no side reaction occurs with cysteine in the protein.

Description

Partially acylated non-natural sugar for metabolic markers and application The application relates to a new bose biotechnology limited company with the application of artificial fertilizer, the application name of which is 'non-natural sugar and a synthetic method and application', and the application number of which is the divisional application of the application patent application with 202211329008.3. Technical Field The invention relates to the technical field of organic chemical synthesis, in particular to partially acylated non-natural sugar for metabolic markers and application thereof. Background The glycosylation modification has important functions and has important significance for observing the glycosylation modification. In cells, metabolic markers using non-natural sugars with chemical reports have been widely used for glycoimaging and glycoprotein group analysis. In vitro, the chemical enzymatic method adds non-natural sugar with chemical report to sugar chain under the action of glycosyltransferase for further study. Unnatural sugars are essential for metabolic and chemoenzymatic labelling. Three main categories are: a. The non-natural sugar without protecting group can be used for metabolic labeling, has high concentration requirement and is a synthetic substrate of non-natural nucleoside sugar used by a chemical enzyme method; b. fully acetylated saccharides have been developed to overcome the poor permeability of non-natural sugar cells without protecting groups, significantly reducing the required concentration of metabolic markers. The existing non-natural sugar probes protected by peracetylation are commercially available and sold by Sigma-Aldrich, CLICK CHEMICAL Tools, etc. However, in 2018, people find that the sulfydryl of the peracetylated saccharide and the protein cysteine generates a non-enzymatic S side reaction for the first time, and a false positive signal is introduced to the application of imaging, mass spectrum identification and the like. C. Partially protected unnatural saccharides after S side reactions have been found, partially hydroxyl acylated unnatural saccharides have evolved, which do not introduce S side reactions while allowing for efficient labeling of sugar metabolism across cell membranes. At this stage, there are mainly two methods for preparing non-natural sugars where the N-acyl position contains azide and the hydroxyl group is not protected. Firstly, synthesizing a sugar derivative with halogen at an N-acyl position, then substituting halogen by azide, and introducing the azide into the N-acyl position of the sugar, secondly, synthesizing azidoacetic acid, and then coupling the azidoacetic acid and amino sugar to obtain the non-natural sugar with the N-acyl position containing the azide. Non-natural saccharides containing other orthogonal groups (e.g., alkynyl groups) and having hydroxy groups not protected are synthesized using the strategy described above for direct coupling of amino sugars to small molecules containing orthogonal groups. However, both synthetic methods have disadvantages in that in the first method, sodium azide is in a large excess and the reaction needs to be heated, so that the risk of explosion exists, and in the second method, the synthesis of the azidoacetic acid takes iodoacetic acid and the excess sodium azide as raw materials, and in order to improve the yield of the azidoacetic acid, the aqueous solution containing the excess sodium azide needs to be acidified and the azidoacetic acid needs to be extracted by an organic solvent, so that the operation has the risk of explosion. Moreover, azidoacetic acid has a low boiling point and is volatile, requires careful handling and can only be synthesized in small quantities. Unprotected unnatural sugars are very polar and often co-elute with the product when chromatographed using a silica gel column. Thus, repeated column chromatography purification is required to obtain sufficiently pure unprotected sugar. There are few reports of purification of unprotected sugars using P-2 gel columns, but this procedure is time consuming, gel columns are expensive and purification of only small amounts of product is possible. Currently, partially protected non-natural sugars fall into two categories, 1, 3-diacylated non-natural sugars and 1, 6-diacylated non-natural sugars. There are several reported examples of 1, 3-bisacylated non-natural sugars, 1, 6-bisacylated non-natural sugars only being 1,6-Pr 2 GalNAz. The synthesis of the 1, 3-double acylated non-natural sugar is to synthesize the non-natural sugar without protecting group, take the non-natural sugar as initial material, protect the No.4 and No.6 positions of the sugar with propylene, acylat the hydroxyl of the No. 1 and No.3 positions, and finally remove the propylene to obtain the 1, 3-double acylated non-natural sugar. In this synthetic route, the above-stated problems are faced when synthesizing a non-natural sugar raw material wi