Ac4ManNAz
(Synonyms: 1,3,4,6-四-O-乙酰基-N-叠氮乙酰基氨基甘露糖) 目录号 : GC60038
Ac4ManNAz是一种含叠氮基的代谢糖蛋白标记试剂，可以选择性修饰蛋白质。Ac4ManNAz是一种点击化学试剂，它含有叠氮基团，可以与含有炔基的分子发生Cu(I)催化的叠氮化物-炔环加成反应（CuAAC）。
Cas No.:361154-30-5
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
建议使用以下通用方案，对GlpBio生产的带有叠氮化物的炔烃修饰寡核苷酸或DNA进行化学标记。本方案仅提供一个指导，请根据您的具体需要进行修改。
- 使用下表计算Click化学标记所需试剂的体积。准备所需的储备溶液（见附录）。
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试剂 |
混合物中的最终浓度 |
原液浓度 |
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炔烃修饰的寡核苷酸或DNA |
Varies (20 — 200 uM) |
Varies |
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叠氮化物 |
1.5 x （寡核苷酸浓度） |
10 mM in DMSO |
|
DMSO |
50 vol % |
— |
|
抗坏血酸 |
0.5 mM |
5 mM in water |
|
铜-TBTA络合物 |
0.5 mM |
10 mM in 55 vol % DMSO |
- 将炔基修饰的寡核苷酸或DNA溶解在水中，置于耐压瓶中。
- 添加2M三乙胺醋酸盐缓冲液（pH 7.0）至最终浓度0.2M。
- 添加DMSO，并涡旋混匀。
- 添加叠氮化物储备溶液（10 mM DMSO中），并涡旋混匀。
- 向混合物中添加所需体积的5mM抗坏血酸储备溶液，并短暂涡旋。
- 通过在溶液中鼓泡惰性气体（如氮气、氩气或氦气）30秒进行脱气。
- 向混合物中添加所需量的10mM铜（II）-TBTA储备溶液（55% DMSO中），用惰性气体冲洗瓶子并关闭瓶盖。
- 充分涡旋混合。如果观察到显著的叠氮化物沉淀，加热瓶子至80°C并涡旋3分钟。
- 在室温下静置过夜。
11. 使用丙酮（用于寡核苷酸）或乙醇（用于DNA）沉淀结合物。充分混合并在−20°C保存20分钟。
对于寡核苷酸结合物的沉淀：向混合物中添加至少4倍体积的3%高氯酸锂丙酮溶液（如果混合物体积较大，分成几个瓶子）。
对于DNA结合物的沉淀：向混合物中添加醋酸钠至最终浓度0.3M；添加2.5倍体积的乙醇（或0.8倍体积的异丙醇）。
- 以10000 rpm离心10分钟。
- 丢弃上清液。用丙酮（1mL）洗涤沉淀物，以10000 rpm离心10分钟。
- 丢弃上清液，干燥沉淀物，并通过反相高效液相色谱（RP-HPLC）或聚丙烯酰胺凝胶电泳（PAGE）纯化结合物。
附录。用于点击化学标记和缀合的试剂储备溶液的制备。
|
5 mM抗坏血酸 |
|
|
准备 |
将18mg抗坏血酸溶解在20mL蒸馏水中。 |
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贮存 |
抗坏血酸很容易被空气氧化。该溶液可稳定一天。请使用新鲜的制剂。 |
|
10 mM铜(II)-TBTA贮存于55% DMSO中 |
|
|
准备 |
将50 mg五水硫酸铜(II)溶解在10 mL蒸馏水中。将116 mg TBTA 配体溶解在11 mL DMSO中。混合两种溶液。 |
|
贮存 |
室温保存。溶液可稳定保存数年。 |
|
2M 三乙基乙酸铵缓冲液，pH 7.0 |
|
|
准备 |
将2.78 mL三乙胺与1.14 mL乙酸混合。加水至10mL 体积，并将pH调节至7.0。 |
|
贮存 |
室温保存。溶液可稳定保存数年。 |
Ac4ManNAz (1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose) is an azide-containing metabolic glycoprotein labeling reagent that can selectively modify proteins for cell labeling, tracking, and proteomic analysis [1, 2]. Ac4ManNAz is a click chemistry reagent that contains an azide group and can undergo Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with molecules containing alkyne groups[3]. Ac4ManNAz can also undergo ring strain-driven alkyne-azide cycloaddition (SPAAC) with molecules containing DBCO or BCN groups[4].
Click chemistry is a versatile reaction that can be used for the synthesis of a variety of conjugates. Virtually any biomolecule can be easily labelled with small molecules, such as fluorescent dyes, biotin, etc using click chemistry method.
Click chemistry reaction takes place between two components: azide and alkyne (terminal acetylene). Both azido and alkyne groups are nearly never encountered in natural biomolecules. Hence, the reaction is highly bioorthogonal and specific.
References:
[1] Han S S, Lee D E, Shim H E, et al. Physiological effects of Ac4ManNAz and optimization of metabolic labeling for cell tracking[J]. Theranostics, 2017, 7(5):1164.
[2] Han S S, Shim H E, Park S J, et al. Safety and optimization of metabolic labeling of endothelial progenitor cells for tracking[J]. Scientific reports, 2018, 8(1): 13212.
[3] Singh M S, Chowdhury S, Koley S. Advances of azide-alkyne cycloaddition-click chemistry over the recent decade[J]. Tetrahedron, 2016, 72(35): 5257-5283.
[4] Vidyakina A A, Silonov S A, Govdi A I, et al. Key role of cycloalkyne nature in alkyne-dye reagents for enhanced specificity of intracellular imaging by bioorthogonal bioconjugation[J]. Organic & Biomolecular Chemistry, 2024.
Ac4ManNAz（1,3,4,6-四-O-乙酰基-N-叠氮乙酰基氨基甘露糖）是一种含叠氮基的代谢糖蛋白标记试剂，可以选择性修饰蛋白质，用于细胞标记、跟踪和蛋白质组学分析[1, 2]。Ac4ManNAz是一种点击化学试剂，它含有叠氮基团，可以与含有炔基的分子发生Cu(I)催化的叠氮化物-炔环加成反应（CuAAC）[3]。Ac4ManNAz还可以和含有 DBCO 或 BCN 基团的分子发生环张力驱动的炔-叠氮环加成反应（SPAAC）[4]。
点击化学是一种多功能反应，可用于合成各种缀合物。事实上，任何生物分子都可以使用点击化学方法轻松地用小分子标记，例如荧光染料、生物素等。
点击化学反应发生在两种组分之间：叠氮化物和炔烃（末端乙炔）。叠氮基和炔基在天然生物分子中几乎从未遇到过。因此，该反应具有高度生物正交性和特异性
| Cas No. | 361154-30-5 | SDF | |
| 别名 | 1,3,4,6-四-O-乙酰基-N-叠氮乙酰基氨基甘露糖 | ||
| Canonical SMILES | O=C(N[C@@H]1C(OC(C)=O)O[C@H](COC(C)=O)[C@@H](OC(C)=O)[C@@H]1OC(C)=O)CN=[N+]=[N-] | ||
| 分子式 | C16H22N4O10 | 分子量 | 430.37 |
| 溶解度 | DMSO:100 mg/mL (232.36 mM; Need ultrasonic) | 储存条件 | Store at 2-8°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
 |
1 mg | 5 mg | 10 mg |
| 1 mM | 2.3236 mL | 11.6179 mL | 23.2358 mL |
| 5 mM | 0.4647 mL | 2.3236 mL | 4.6472 mL |
| 10 mM | 0.2324 mL | 1.1618 mL | 2.3236 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Physiological Effects of Ac4ManNAz and Optimization of Metabolic Labeling for Cell Tracking
Theranostics 2017 Mar 1;7(5):1164-1176.PMID:28435456DOI:10.7150/thno.17711.
Metabolic labeling techniques are powerful tools for cell labeling, tracking and proteomic analysis. However, at present, the effects of the metabolic labeling agents on cell metabolism and physiology are not known. To address this question, in this study, we analyzed the effects of cells treated with Ac4ManNAz through microarray analysis and analyses of membrane channel activity, individual bio-physiological properties, and glycolytic flux. According to the results, treatment with 50 μM Ac4ManNAz led to the reduction of major cellular functions, including energy generation capacity, cellular infiltration ability and channel activity. Interestingly, 10 μM Ac4ManNAz showed the least effect on cellular systems and had a sufficient labeling efficiency for cell labeling, tracking and proteomic analysis. Based on our results, we suggest 10 μM as the optimum concentration of Ac4ManNAz for in vivo cell labeling and tracking. Additionally, we expect that our approach could be used for cell-based therapy for monitoring the efficacy of molecule delivery and the fate of recipient cells.
Safety and Optimization of Metabolic Labeling of Endothelial Progenitor Cells for Tracking
Sci Rep 2018 Sep 4;8(1):13212.PMID:30181604DOI:10.1038/s41598-018-31594-0.
Metabolic labeling is one of the most powerful methods to label the live cell for in vitro and in vivo tracking. However, the cellular mechanisms by modified glycosylation due to metabolic agents are not fully understood. Therefore, metabolic labeling has not yet been widely used in EPC tracking and labeling. In this study, cell functional properties such as proliferation, migration and permeability and gene expression patterns of metabolic labeling agent-treated hUCB-EPCs were analyzed to demonstrate cellular effects of metabolic labeling agents. As the results, 10 μM Ac4ManNAz treatment had no effects on cellular function or gene regulations, however, higher concentration of Ac4ManNAz (>20 μM) led to the inhibition of functional properties (proliferation rate, viability and rate of endocytosis) and down-regulation of genes related to cell adhesion, PI3K/AKT, FGF and EGFR signaling pathways. Interestingly, the new blood vessel formation and angiogenic potential of hUCB-EPCs were not affected by Ac4ManNAz concentration. Based on our results, we suggest 10 μM as the optimal concentration of Ac4ManNAz for in vivo hUCB-EPC labeling and tracking. Additionally, we expect that our approach can be used for understanding the efficacy and safety of stem cell-based therapy in vivo.
Bio-orthogonal click-targeting nanocomposites for chemo-photothermal synergistic therapy in breast cancer
Theranostics 2020 Apr 6;10(12):5305-5321.PMID:32373214DOI:10.7150/thno.42445.
Chemo-photothermal synergistic treatment has a high potential to complement traditional cancer therapy and amplify its outcome. Precision in the delivery of these therapeutic agents to tumor cells has been indicated as being key to maximizing their therapeutic effects. Method: We developed a bio-orthogonal copper-free click-targeting nanocomposite system (DLQ/DZ) that markedly improved specific co-delivery of the chemotherapeutic agent doxorubicin and the photosensitizer zinc phthalocyanine to breast cancer cells via a two-step mechanism. In the first step, an azide-modified sugar (tetraacetylated N-azidoacetyl-D-mannosamine, Ac4ManNAz) was injected intratumorally for glycoengineering of the tumor cell surface. Subsequently, DLQ/DZ was administered to achieve tumor enrichment via bio-orthogonal copper-free click-targeting. Results: During the first step in our experiments, high density azide groups (3.23×107/cell) were successfully glycoengineered on the surface of tumor cells following Ac4ManNAz administration in vitro. Subsequently, the highly efficient bio-orthogonal click chemical reaction between receptor-like azide groups on tumor cells and DBCO on nanocomposites significantly enhanced the cellular uptake and tumor-specific distribution (4.6x increase) of the nanocomposites in vivo. Importantly, Ac4ManNAz+DLQ/DZ treatment augmented the anti-cancer effect of combined chemotherapy and PTT (96.1% inhibition rate), nearly ablating the tumor. Conclusions: This bio-orthogonal click-targeting combination strategy may provide a promising treatment approach for surficial breast cancers.
Mass spectrometric analysis of products of metabolic glycan engineering with azido-modification of sialic acids
Anal Bioanal Chem 2015 Dec;407(30):8945-58.PMID:26362153DOI:10.1007/s00216-015-9010-x.
Metabolic engineering of glycans present on antibodies and other glycoproteins is becoming an interesting research area for improving our understanding of the glycome. With knowledge of the sialic acid biosynthetic pathways, the experiments described in this report are based on a published procedure involving the addition of a synthesized azido-mannosamine sugar into cell culture media and evaluation of downstream expression as azido-sialic acid. This unique bioorthogonal sugar has the potential for a variety of "click chemistry" reactions through the azide linkage, which allow for it to be isolated and quantified given the choice of label. In this report, mass spectrometry was used to investigate and optimize the cellular absorption of peracetylated N-azidoacetylmannosamine (Ac4ManNAz) to form N-azidoacetylneuraminic acid (SiaNAz) in a Chinese hamster ovary (CHO) cell line transiently expressing a double mutant trastuzumab (TZMm2), human galactosyltransferase 1 (GT), and human α-2,6-sialyltransferase (ST6). This in vivo approach is compared to in vitro enzymatic addition SiaNAz onto TZMm2 using soluble β-galactosamide α-2,6-sialyltransferase 1 and CMP-SiaNAz as donor. The in vivo results suggest that for this mAb, concentrations above 100 μM of Ac4ManNAz are necessary to allow for observation of terminal SiaNAz on tryptic peptides of TZMm2 by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. This is further confirmed by a parallel study on the production of EG2-hFc monoclonal antibody (Zhang J et al. Prot Expr Purific 65(1); 77-82, 2009) in the presence of increasing concentrations of Ac4ManNAz.
Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes In Vivo
Bioconjug Chem 2016 Apr 20;27(4):927-36.PMID:26930274DOI:10.1021/acs.bioconjchem.6b00010.
Establishment of an appropriate cell labeling and tracking method is essential for the development of cell-based therapeutic strategies. Here, we are introducing a new method for cell labeling and tracking by combining metabolic gylcoengineering and bioorthogonal copper-free Click chemistry. First, chondrocytes were treated with tetraacetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz) to generate unnatural azide groups (-N3) on the surface of the cells. Subsequently, the unnatural azide groups on the cell surface were specifically conjugated with near-infrared fluorescent (NIRF) dye-tagged dibenzyl cyclooctyne (DBCO-650) through bioorthogonal copper-free Click chemistry. Importantly, DBCO-650-labeled chondrocytes presented strong NIRF signals with relatively low cytotoxicity and the amounts of azide groups and DBCO-650 could be easily controlled by feeding different amounts of Ac4ManNAz and DBCO-650 to the cell culture system. For the in vivo cell tracking, DBCO-650-labeled chondrocytes (1 × 10(6) cells) seeded on the 3D scaffold were subcutaneously implanted into mice and the transplanted DBCO-650-labeled chondrocytes could be effectively tracked in the prolonged time period of 4 weeks using NIRF imaging technology. Furthermore, this new cell labeling and tracking technology had minimal effect on cartilage formation in vivo.