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Peptide Ac-Arg-Leu-Arg-MCA is a Research Peptide with significant interest within the field academic and medical research. Recent citations using Ac-Arg-Leu-Arg-MCA include the following: Collagen-like polypeptide poly (Pro-Hyp-Gly) conjugated with Gly-Arg-Gly-Asp-Ser and Pro-His-Ser-Arg-Asn peptides enhances cell adhesion, migration, and Y Shibasaki, S Hirohara , K Terada, T Ando - Peptide , 2011 - Wiley Online Libraryhttps://onlinelibrary.wiley.com/doi/abs/10.1002/bip.21551 Synthesis, nano-scale assembly, and in vivo anti-thrombotic activity of novel short peptides containing L-Arg and L-Asp or L-Glu Y Chen, G Cui, M Zhao, C Wang, K Qian - Bioorganic & medicinal , 2008 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S0968089608003921 Possible existence of common internalization mechanisms among arginine-rich peptides T Suzuki, S Futaki, M Niwa, S Tanaka, K Ueda - Journal of Biological , 2002 - ASBMBhttps://www.jbc.org/article/S0021-9258(20)87688-1/abstract Selective cytotoxicity following Arg-to-Lys substitution in tritrpticin adopting a unique amphipathic turn structure ST Yang, SY Shin, CW Lee, YC Kim , KS Hahm, JI Kim - FEBS letters, 2003 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S0014579303002667 Relevance of the C-terminal Arg-Phe sequence in γ2-melanocyte-stimulating hormone (γ2-MSH) for inducing cardiovascular effects in conscious rats M Nijsen, GJW De Ruiter - British journal of , 2000 - Wiley Online Libraryhttps://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1038/sj.bjp.0703709 Improving the activity of Trp-rich antimicrobial peptides by Arg/Lys substitutions and changing the length of cationic residues M Arias , KB Piga, ME Hyndman, HJ Vogel - Biomolecules, 2018 - mdpi.comhttps://www.mdpi.com/2218-273X/8/2/19 Research progress of radiolabeled Asn-Gly-Arg (NGR) peptides for imaging and therapy L Zhu, Z Ding, X Li, H Wei, Y Chen - Molecular Imaging, 2020 - journals.sagepub.comhttps://journals.sagepub.com/doi/abs/10.1177/1536012120934957 Substitution of Gly with Ala enhanced the melanoma uptake of technetium-99m-labeled Arg-Ala-Asp-conjugated alpha-melanocyte stimulating hormone peptide J Yang, Y Miao - Bioorganic & medicinal chemistry letters, 2012 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S0960894X12000169 resulting in improved melanoma uptake and reduced renal uptake of Tc-99m-labeled Arg-Gly-Asp-conjugated alpha-melanocyte stimulating hormone hybrid peptide J Yang, H Guo , RS Padilla, M Berwick - Bioorganic & medicinal , 2010 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S096808961000725X Dual receptor-targeting 99mTc-labeled Arg-Gly-Asp-conjugated Alpha-Melanocyte stimulating hormone hybrid peptides for human melanoma imaging J Xu , J Yang, Y Miao - Nuclear medicine and biology, 2015 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S0969805114005356 Highly active antibacterial ferrocenoylated or ruthenocenoylated Arg-Trp peptides can be discovered by an L-to-D substitution scan HB Albada , P Prochnow, S Bobersky , JE Bandow - Chemical , 2014 - pubs.rsc.orghttps://pubs.rsc.org/en/content/articlehtml/2014/sc/c4sc01822b A bioconjugate of Lys and Arg mimics biological activity of the Arg-Gly-Asp peptide sequence G Ehteshami, DC Brune, JC Lopez, SP Massia - Acta Biomaterialia, 2005 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S1742706104000108 Evaluation of new Tc-99m-labeled Arg-X-Asp-conjugated alpha-melanocyte stimulating hormone peptides for melanoma imaging AM Flook, J Yang, Y Miao - Molecular pharmaceutics, 2013 - ACS Publicationshttps://pubs.acs.org/doi/abs/10.1021/mp400248f Linker Dramatically Decreased the Renal Uptake of 99mTc-Labeled Arg-X-Asp-Conjugated and X-Ala-Asp-Conjugated alpha-Melanocyte Stimulating Hormone Peptides AM Flook, J Yang, Y Miao - Journal of medicinal chemistry, 2014 - ACS Publicationshttps://pubs.acs.org/doi/abs/10.1021/jm501114v Effects of amino acids on melanoma targeting and clearance properties of Tc-99m-labeled Arg-X-Asp-conjugated alpha-melanocyte stimulating hormone peptides AM Flook, J Yang, Y Miao - Journal of medicinal chemistry, 2013 - ACS Publicationshttps://pubs.acs.org/doi/abs/10.1021/jm4012356

Fluorogenic substrate peptide to assay trypsin-like activity. In its intact state this peptide is non-fluorescent, however when aminomethylcoumarin (AMC) is released upon hydrolysation, fluorescence can be detected. This peptide is therefore a useful tool for analysing trypsin-like enzyme activity.AMC is a fluorescent dye with excitation maxima at around 360 nm and emission maxima at around 450 nm. AMC can be excited with a mercury lamp and observed using a UV filter set.

Ac-Arg-Leu-Arg-AMC trifluoroacetate salt is a mitochondrial biogenesis activator that has been shown to increase the levels of proteins in the mitochondria. These proteins are required for mitochondrial membrane potential, ATP production, and protein homeostasis. Ac-Arg-Leu-Arg-AMC trifluoroacetate salt has been shown to increase the number of pluripotency markers in human liver cells and to reduce insulin resistance in animals. The drug also increases the expression of ubiquitin ligases and proteasomes, which are enzymes that degrade damaged proteins. Ac-Arg-Leu-Arg-AMC trifluoroacetate salt may be used for treating liver diseases or disorders as well as obesity.

Ac-Arg-Leu-Arg-MCA is a fluorogenic substrate for the proteasome that can be used in proteasome research. Ac-Arg-Leu-Arg-MCA has been shown to be a useful fluorescent substrate for the ubiquitin proteasome system substrates and peptides and biochemicals. It is also an Enzyme Substrate of the Peptides & Biochemicals section.

7-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。

7-Amino-4-methyl-coumaride (AMC or Amc) is fluorophor with an excitation at 350 nm  and emission of 450 nm .

Ac-RLR-AMC, fluorogenic substrate for assaying the trypsin-like activity of purified proteasomes (Km = 78 μM).

多肽Ac-Arg-Leu-Arg-AMC的合成步骤：

1、合成CTC树脂：称取2.87g CTC Resin（如初始取代度约为0.86mmol/g）和2.96mmol Fmoc-Arg(Pbf)-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7.4mmol 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-Arg(Pbf)-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-Arg(Pbf)-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7.4mmol Fmoc-Leu-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14.81mmol DIPEA，7.03mmol 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-Leu-Arg(Pbf)-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-Leu-Arg(Pbf)-CTC Resin

Fmoc-Arg(Pbf)-Leu-Arg(Pbf)-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-Arg(Pbf)-Leu-Arg(Pbf)-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7.4mmol 乙酸酐到树脂中，再加入14.81mmol DIPEA、7.03mmol 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-Arg(Pbf)-Leu-Arg(Pbf)-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-Arg(Pbf)-Leu-Arg(Pbf)-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-Arg(Pbf)-Leu-Arg(Pbf)-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7.4mmol 7-氨基-4-甲基香豆素到树脂中，再加入14.81mmol DIPEA、7.03mmol 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-Arg(Pbf)-Leu-Arg(Pbf)-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-Arg-Leu-Arg-AMC。结构图见产品结构图。

切割液选择：1）TFA：H2O=95%：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

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