400-998-5282
专注多肽 服务科研
400-998-5282
专注多肽 服务科研
A continuous fluorometric assay employing Ac-DEVD-AMC has been developed for the measurement of caspase-3, a cysteine protease also called apopain, Yama or CPP-32. The design of this substrate Ac-DEVD-AMC was based on the substrate motif that has been use
编号：145638
CAS号：169332-61-0
单字母：Ac-DEVD-AMC
| 编号: | 145638 |
| 中文名称: | Caspase 3 (Apopain) Substrate 1m, fluorogenic |
| 英文名: | Caspase 3 (Apopain) Substrate 1m, fluorogenic |
| 英文同义词: | Apopain Substrate |
| CAS号: | 169332-61-0 |
| 单字母: | Ac-DEVD-AMC |
| 三字母: | Ac N端乙酰化封端，一种常见的修饰方式，常用于模拟蛋白质中的肽片段。 -AspL-天冬氨酸：aspartic acid。系统命名为(2S)-氨基-丁二酸。是编码氨基酸，又是神经递质。符号：D，Asp。D-天冬氨酸存在于多种细菌的细胞壁和短杆菌肽A中。 -GluL-谷氨酸：glutamic acid。系统命名为(2S)-氨基-戊二酸。是编码氨基酸。符号：E，Glu。D-谷氨酸存在于多种细菌的细胞壁和某些细菌杆菌肽中。 -ValL-缬氨酸：valine。系统命名为(2S)-氨基-3-甲基丁酸。是编码氨基酸。是哺乳动物的必需氨基酸。符号：V，Val。在某些放线菌素如缬霉素中存在 D-缬氨酸。 -AspL-天冬氨酸：aspartic acid。系统命名为(2S)-氨基-丁二酸。是编码氨基酸，又是神经递质。符号：D，Asp。D-天冬氨酸存在于多种细菌的细胞壁和短杆菌肽A中。 -AMC7-氨基-4-甲基香豆素（AMC或Amc）是一种荧光染料，其激发波长为350纳米，发射波长为450纳米。 |
| 氨基酸个数: | 4 |
| 分子式: | C30H37O13N5 |
| 平均分子量: | 675.64 |
| 精确分子量: | 675.24 |
| 等电点(PI): | - |
| pH=7.0时的净电荷数: | -3 |
| 平均亲水性: | 1.875 |
| 疏水性值: | -1.58 |
| 外观与性状: | 白色粉末状固体 |
| 消光系数: | - |
| 来源: | 人工化学合成，仅限科学研究使用，不得用于人体。 |
| 纯度: | 95%、98% |
| 盐体系: | 可选TFA、HAc、HCl或其它 |
| 生成周期: | 2-3周 |
| 储存条件: | 负80℃至负20℃ |
| 标签: | AMC修饰肽 |
A continuous fluorometric assay employing Ac-DEVD-AMC has been developed for the measurement of caspase-3, a cysteine protease also called apopain, Yama or CPP-32. The design of this substrate Ac-DEVD-AMC was based on the substrate motif that has been used successfully with caspase-1, using the poly(ADP-ribose) polymerase cleavage site P₁-P₄ tetrapeptide.
Ac-Tyr-Val-Gly is a mitochondrial protein that regulates mitochondrial functions and is involved in the regulation of apoptosis. Ac-Tyr-Val-Gly interacts with nuclear DNA and regulates transcription, translation, and replication. Ac-Tyr-Val-Gly has been shown to be toxic to liver cells; however, it has been shown to have no effect on neuronal death or apoptosis pathway. These effects may be due to its ability to induce proteolytic activity in neurons and its ability to activate proapoptotic proteins such as Bax.
Ac-Asp-Glu-Val-Asp-AMC is an ion channel activator. It binds to the receptor site of the ligand, which causes a conformational change in the protein that leads to the opening of the ion channel. Ac-Asp-Glu-Val-Asp-AMC is a peptide with a molecular weight of 927.6 and is soluble in water. This product has been shown to be stable under various conditions and can be used as research tool or pharmacological agent. Ac-Asp-Glu-Val-Asp-AMC is also an antibody that binds to an epitope on human CD8 protein and can be used for cell biology studies, such as activation and inhibition of these proteins.
Ac-Asp-Glu-Val-Asp-AMC ammonium salt is a small molecule that is used as a tool to study apoptosis in vitro. Ac-Asp-Glu-Val-Asp-AMC ammonium salt induces apoptosis by blocking the mitochondrial membrane potential and inducing the release of cytochrome c from mitochondria into the cytoplasm. This drug also induces activation of caspase 3, which initiates the cascade of events leading to cell death. Ac-Asp-Glu-Val-Asp-AMC ammonium salt has been shown to have an antiangiogenic effect on hl60 cells. This effect may be due to its ability to inhibit expression of survivin, a protein that protects cells from apoptosis. The efficacy of this drug in an experimental model has been shown to be dependent on toll like receptor (TLR) signaling pathways and mitochondrial function.br>
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多肽Ac-Asp-Glu-Val-Asp-AMC的合成步骤：
1、合成CTC树脂：称取2.97g CTC Resin（如初始取代度约为0.41mmol/g）和1.46mmol Fmoc-Asp(OtBu)-OH于反应器中，加入适量DCM溶解氨基酸（需要注意，此时CTC树脂体积会增大好几倍，避免DCM溶液过少），再加入3.65mmol DIPEA（Mw：129.1,d：0.740g/ml），反应2-3小时后，可不抽滤溶液，直接加入1ml的HPLC级甲醇，封端半小时。依次用DMF洗涤2次，甲醇洗涤1次，DCM洗涤一次，甲醇洗涤一次，DCM洗涤一次，DMF洗涤2次（这里使用甲醇和DCM交替洗涤，是为了更好地去除其他溶质，有利于后续反应）。得到 Fmoc-Asp(OtBu)-CTC Resin。结构图如下：
2、脱Fmoc：加3倍树脂体积的20%Pip/DMF溶液，鼓氮气30分钟，然后2倍树脂体积的DMF 洗涤5次。得到 H2N-Asp(OtBu)-CTC Resin 。（此步骤脱除Fmoc基团，茚三酮检测为蓝色，Pip为哌啶）。结构图如下：
3、缩合：取3.65mmol Fmoc-Val-OH 氨基酸，加入到上述树脂里，加适当DMF溶解氨基酸，再依次加入7.31mmol DIPEA，3.47mmol HBTU。反应30分钟后，取小样洗涤，茚三酮检测为无色。用2倍树脂体积的DMF 洗涤3次树脂。（洗涤树脂，去掉残留溶剂，为下一步反应做准备）。得到Fmoc-Val-Asp(OtBu)-CTC Resin。氨基酸：DIPEA：HBTU：树脂=3：6：2.85：1（摩尔比）。结构图如下：
4、依次循环步骤二、步骤三，依次得到
H2N-Val-Asp(OtBu)-CTC Resin
Fmoc-Glu(OtBu)-Val-Asp(OtBu)-CTC Resin
H2N-Glu(OtBu)-Val-Asp(OtBu)-CTC Resin
Fmoc-Asp(OtBu)-Glu(OtBu)-Val-Asp(OtBu)-CTC Resin
以上中间结构，均可在专肽生物多肽计算器-多肽结构计算器中，一键画出。
最后再经过步骤二得到 H2N-Asp(OtBu)-Glu(OtBu)-Val-Asp(OtBu)-CTC Resin，结构如下：
5、乙酸酐反应连接：在上述树脂中，加入适当DMF后，再加入3.65mmol 乙酸酐到树脂中，再加入7.31mmol DIPEA、3.47mmol HBTU，鼓氮气反应30分钟。用2倍树脂体积的DMF 洗涤3次树脂（洗涤树脂，去掉残留溶剂，为下一步反应做准备）。 得到Ac-Asp(OtBu)-Glu(OtBu)-Val-Asp(OtBu)-CTC Resin。 结构如下：
6、全保护切割：配置0.5%TFA/DCM溶液，溶液体积约为树脂体积的3倍。再次用DCM洗涤树脂2遍（去除残留DMF），后将配置好的溶液倒入到反应器中，反应30分钟。抽滤树脂，收集滤液（此时多肽已经从树脂上分离，存在于滤液中）。多肽序列为 Ac-Asp(OtBu)-Glu(OtBu)-Val-Asp(OtBu)-CTC Resin。 在滤液中添加DIEPA，调PH至7-8。用饱和NaHCO3洗涤滤液，分离出DCM层溶液。可适当旋蒸DCM层溶液，减少有机溶剂。再次加入1或2倍体积的乙酸乙酯，用稀HCl溶液调PH至微酸性，将多肽从DCM层萃取到乙酸乙酯层。用饱和NaCl洗涤2次乙酸乙酯层。用无水硫酸镁吸收乙酸乙酯层的水分。通过减压旋蒸，直接将乙酸乙酯完全旋蒸掉，得到晶体状固体多肽，用于下一步C端反应。或通过减压旋蒸保留适量乙酸乙酯的溶液体积，加入冰乙醚析出 多肽，然后对多肽进行烘干操作即可用于下一步C端反应。Ac-Asp(OtBu)-Glu(OtBu)-Val-Asp(OtBu)-COOH的结构图如下。
7、7-氨基-4-甲基香豆素反应连接：在上述树脂中，加入适当DMF后，再加入3.65mmol 7-氨基-4-甲基香豆素到树脂中，再加入7.31mmol DIPEA、3.47mmol HBTU，鼓氮气反应30分钟。用2倍树脂体积的DMF 洗涤3次树脂（洗涤树脂，去掉残留溶剂，为下一步反应做准备）。 得到 Ac-Asp(OtBu)-Glu(OtBu)-Val-Asp(OtBu)-AMC。 结构如下：
8、切割：6倍树脂体积的切割液（或每1g树脂加8ml左右的切割液），摇床摇晃 2小时，过滤掉树脂，用冰无水乙醚沉淀滤液，并用冰无水乙醚洗涤沉淀物3次，最后将沉淀物放真空干燥釜中，常温干燥24小试，得到粗品Ac-Asp-Glu-Val-Asp-AMC。结构图见产品结构图。
切割液选择：1）TFA：H2O=95%：5%、TFA：H2O=97.5%：2.5%
2）TFA：H2O：TIS=95%：2.5%：2.5%
3）三氟乙酸：茴香硫醚：1，2-乙二硫醇：苯酚：水=87.5%：5%：2.5%：2.5%：2.5%
（前两种适合没有容易氧化的氨基酸，例如Trp、Cys、Met。第三种适合几乎所有的序列。）
9、纯化冻干：使用液相色谱纯化，收集目标峰液体，进行冻干，获得蓬松的粉末状固体多肽。不过这时要取小样复测下纯度 是否目标纯度。
10、最后总结：
杭州专肽生物技术有限公司（ALLPEPTIDE https://www.allpeptide.com）主营定制多肽合成业务，提供各类长肽，短肽，环肽，提供各类修饰肽，如：荧光标记修饰（CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等），功能基团修饰肽（叠氮、炔基、DBCO、DOTA、NOTA等），同位素标记肽（N15、C13），订书肽（Stapled Peptide）,脂肪酸修饰肽（Pal、Myr、Ste），磷酸化修饰肽（P-Ser、P-Thr、P-Tyr），环肽（酰胺键环肽、一对或者多对二硫键环），生物素标记肽，PEG修饰肽，甲基化修饰肽
以上所有内容，为专肽生物原创内容，请勿发布到其他网站上。
