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Glycometabolism labeling probe, kit containing same and application thereof

A technology for labeling probes and sugar metabolism, applied in biological testing, microbial determination/inspection, instruments, etc. effect of reaction

Active Publication Date: 2021-08-24
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, unprotected sugar probes require millimolar concentrations to effectively label cells, which greatly limits the application of unnatural sugar metabolism markers.
Therefore, the existing non-natural sugar probes protected by full acetylation have been commercialized at this stage, and are sold in companies such as Sigma-Aldrich and Click Chemical Tools, while unprotected non-natural sugar probes have low metabolic efficiency. At present, major chemical reagent companies have not sold
[0009] It can be seen that the existing technology adopts the strategy of full acetylation protection to improve metabolic efficiency but produces cysteine ​​side reactions, while sugar probes without acetylation protection can avoid side reactions of cysteine, but Greatly reduces the metabolic efficiency of the probe

Method used

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  • Glycometabolism labeling probe, kit containing same and application thereof
  • Glycometabolism labeling probe, kit containing same and application thereof
  • Glycometabolism labeling probe, kit containing same and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: 1,3-Ac 2 GalNAz and 1,3-Pr 2 Synthesis of GalNAz

[0069] (A)1,3-Ac 2 The synthesis of GalNAz sees following route (I):

[0070]

[0071] Among them, the conditions of steps a, b and c are respectively:

[0072] Under ice bath, GalNAz (Compound 1, 200 mg, 0.76 mmol) was suspended in 5 mL of acetone, 2,2-dimethoxypropane (0.94 mL, 763 mmol) was added dropwise, and finally camphorsulfonic acid (17.7 mg , 7.63 mmol). The system was slowly raised to 4°C, and stirring was continued for 1h. The reaction was quenched with triethylamine, the solvent was spin-dried, and purified with a silica gel column (ethyl acetate:petroleum ether rose from 1:1 to 4:1) to obtain a white crude product (R f =0.2, ethyl acetate). The product was further purified by HPLC to give a white product (110 mg, 48%, α / β=3:1).

[0073] Its NMR test data are: 1 H NMR (500MHz, CD 3 OD)δ5.23(d,J=3.5Hz,1H),4.67(d,J=8.5Hz,1H),4.33(dd,J=11.0,3.5Hz,1H),4.28(d,J=4.0, 1H),4.24-4.16(m,3H),4...

Embodiment 2

[0088] Example 2 1,3-Ac 2 GalNAz and 1,3-Pr 2 In vitro reaction of GalNAz with protein

[0089] In order to verify 1,3-Ac 2 GalNAz and 1,3-Pr 2 GalNAz cannot spontaneously react with protein cysteine. With unprotected GalNAz as a positive control and fully protected Ac4GalNAz as a negative control, the above two probes were mixed with different cell protein lysates (cell lysates) or single protein After incubation at 37° C. for 2 h, the azide signal on the protein was detected by electrophoresis gel.

[0090] The result is as follows Figure 2a to Figure 2e As shown, among them, Figure 2a Shown is 1,3-Ac 2 GalNAz and 1,3-Pr 2 The detection results of the reaction of two GalNAz probes with the protein lysate of Hela cells; Figure 2b Shown is 1,3-Ac 2 GalNAz and 1,3-Pr 2 The detection results of the reaction of two GalNAz probes to the protein lysate of 293T cells; Figure 2c Shown is 1,3-Ac 2 GalNAz and 1,3-Pr 2 The detection results of GalNAz two probes reacting t...

Embodiment 3

[0092] Example 3 In vivo metabolic labeling effect

[0093] In order to verify its metabolic labeling effect in vivo, the above-mentioned 1,3-Ac 2 GalNAz and 1,3-Pr 2 GalNAz probes for metabolic labeling of HeLa cells. The specific operation steps are:

[0094] 100μM, 200μM, 1mM 1,3-Ac 2 GalNAz, 100 μM, 200 μM 1,3-Ac 2 GalNAz1,3-Pr 2 GalNAz and 200μM and 1mM GalNAz probes were added to the culture medium of Hela cells for culture, and then through bio-orthogonal reaction with Cy5 fluorescently labeled substances, the in-gel fluorescence detection was performed on HeLa cells after different treatments, and the detection results See image 3 ( image 3 CBB in indicates Coomassie Brilliant Blue staining to show loading control).

[0095] From image 3 It can be seen that 100 μM 1,3-Pr 2 The metabolic labeling efficiency of GalNAz is equivalent to or even exceeds the labeling effect of 1mM GalNAz (while Ac 4 GalNAz labeling has side effects, due to false positive labeli...

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Abstract

The invention provides a sugar metabolism labeling probe, a kit containing it and applications thereof. The sugar metabolism labeling probe is a non-natural sugar metabolism labeling probe with partially protected hydroxyl groups. The non-natural sugar metabolism labeling probe with partially protected hydroxyl groups in this application takes into account the advantages of existing probes, not only ensuring that the probe can be efficiently used by cells, but also effectively avoiding the interaction between the probe and the protein in the process of metabolism. Side effects of cysteine.

Description

technical field [0001] The invention relates to the field of biological living body labeling, in particular to a sugar metabolism labeling probe, a kit containing the same and an application thereof. Background technique [0002] Unlike proteins encoded by nucleic acids, the synthesis of glycosylation is a template-free process. Therefore, the labeling of glycosylation cannot be done directly through gene coding, but the cells can be metabolically marked by monosaccharide analogues, and then fluorescent labeling or biotin labeling of specific glycosylation can be performed by using bioorthogonal reactions. [0003] In order to improve the efficiency of probe uptake by cells in traditional non-natural carbohydrate metabolism probes, all hydroxyl groups are protected by acetylation. However, it has also been reported in the prior art that non-natural sugar probes protected by full acetylation can react spontaneously with cysteine ​​on proteins, resulting in a large number of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/50C12Q1/02
CPCG01N33/5005
Inventor 陈兴范欣琦
Owner PEKING UNIV