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Kemptide是一种合成的七肽，作为cAMP依赖性蛋白激酶 (PKA) 的特异性底物。
Kemptide is a synthetic heptapeptide that acts as a specific substrate for cAMP-dependent protein kinase (PKA).

"Peptide Kemptide is a Research Peptide with significant interest within the field academic and medical research. Recent citations using Kemptide include the following: The peptide microarray-based assay for kinase functionality and inhibition study Z Wang - Peptide Microarrays: Methods and Protocols, 2009 - Springerhttps://link.springer.com/protocol/10.1007/978-1-60327-394-7_18 42 Peptide-based affinity labeling of adenosine cyclic monophosphate-dependent protein kinase WT Miller - Methods in enzymology, 1991 - Elsevierhttps://www.sciencedirect.com/science/article/pii/007668799100166T From zyxwvutsrqponmlkjihgf W Merlevede, JR Vandenheede - academia.eduhttps://www.academia.edu/download/45777775/Van_Lint_J_Agostinis_P_Vandevoorde_V_et_20160519-19186-v9i2kw.pdf Relationship of phosphorylation and ADP-ribosylation using a synthetic peptide as a model substrate. SV Kharadia, DJ Graves - Journal of Biological Chemistry, 1987 - ASBMBhttps://www.jbc.org/article/S0021-9258(18)45389-6/abstract Tandem mass spectrometry investigation of ADP-ribosylated kemptide SM Hengel, SA Shaffer , BL Nunn - Journal of the American , 2011 - ACS Publicationshttps://pubs.acs.org/doi/abs/10.1016/j.jasms.2008.10.025 Microarrays of peptides elevated on the protein layer for efficient protein kinase assay SJ Lee , SY Lee - Analytical biochemistry, 2004 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S0003269704003264 Elution-modified displacement chromatography coupled with electrospray ionization-MS: on-line detection of trace peptides at low-femtomole level in peptide digests R Xiang, C Horvath, JA Wilkins - Analytical chemistry, 2003 - ACS Publicationshttps://pubs.acs.org/doi/abs/10.1021/ac026232t Enabling resolution of isomeric peptides using tri-state ion gating and Fourier-transform ion mobility spectrometry P Kwantwi-Barima , T Reinecke , BH Clowers - International Journal for Ion , 2020 - Springerhttps://link.springer.com/article/10.1007/s12127-020-00261-4 Fluram-kemptide-Lys8 non-radioactive assay for protein kinase A NA Araujo , A Guevara, MA Lorenzo , M Calabokis - The Protein , 2016 - Springerhttps://link.springer.com/article/10.1007/s10930-016-9667-9 MALDI-TOF mass-spectrometry-based versatile method for the characterization of protein kinases N Kondo, SI Nishimura - Chemistry-A European Journal, 2009 - Wiley Online Libraryhttps://chemistry-europe.onlinelibrary.wiley.com/doi/abs/10.1002/chem.200801650 Structural basis for peptide binding in protein kinase A: role of glutamic acid 203 and tyrosine 204 in the peptide-positioning loop MJ Moore, JA Adams , SS Taylor - Journal of Biological Chemistry, 2003 - ASBMBhttps://www.jbc.org/article/S0021-9258(19)32548-7/abstract Establishment and Optimization of Protein Labeling Based on Rare Earth Metal Chelated Tags MI Wei, W Jing, Y Wan-Tao, CAI Yun - Chinese Journal of , 2010 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S1872204009600700 Peptide-based substrate and inhibitor studies of serine/threonine protein kinases ME Koszelak - 1998 - search.proquest.comhttps://search.proquest.com/openview/a972f8e48b37621413f8f97d4e2eede0/1?pq-origsite=gscholar&cbl=18750&diss=y Evaluation of kemptide, a synthetic serinecontaining heptapeptide, as a phosphate acceptor for the estimation of cyclic AMP-dependent protein kinase activity in MA Giembycz , J Diamond - Biochemical pharmacology, 1990 - Elsevierhttps://www.sciencedirect.com/science/article/pii/000629529090026H An affinity label of absolute peptidic origin M Prorok , DS Lawrence - Journal of the American Chemical , 1990 - ACS Publicationshttps://pubs.acs.org/doi/pdf/10.1021/ja00179a081 An ether-based peptide analog as c-AMP-dependent protein kinase inhibitor/substrate M Ho, A Uhrin , M Huang - Bioorganic & Medicinal Chemistry Letters, 1994 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S0960894X01801530 Phosphorylation sites within alpha4 subunits of alpha4beta2 neuronal nicotinic receptors: a comparison of substrate specificities for cAMP-dependent protein kinase (PKA) and L Wecker , CQ Rogers - Neurochemical research, 2003 - Springerhttps://link.springer.com/article/10.1023/A:1022892400362 Study of the affinity between the protein kinase PKA and peptide substrates derived from kemptide using molecular dynamics simulations and MM/GBSA K Mena-Ulecia , A Vergara-Jaque , H Poblete - PLoS , 2014 - journals.plos.orghttps://journals.plos.org/plosone/article?id=10.1371/journal.pone.0109639 Study of the affinity between the protein kinase PKA and homoarginine-containing peptides derived from kemptide: Free energy perturbation (FEP) calculations K Mena-Ulecia - Journal of , 2018 - Wiley Online Libraryhttps://onlinelibrary.wiley.com/doi/abs/10.1002/jcc.25176 Use of synthetic peptide libraries and phosphopeptide-selective mass spectrometry to probe protein kinase substrate specificity. JH Till, RS Annan , SA Carr , WT Miller - Journal of Biological Chemistry, 1994 - ASBMBhttps://www.jbc.org/article/S0021-9258(17)37302-7/abstract Nelson A. Araujo, Alberto Guevara, MaracaÂ\xada A. Lorenzo, Maritza Calabokis & J Bubis - academia.eduhttps://www.academia.edu/download/55404331/Araujo_NA__Guevara_A__Lorenzo_M__Calabokis_M__Bubis_J__The_Protein_Journal_2016_version_online.pdf kinase and MAPKAP kinase-1 identifies a relatively specific substrate for p70 S6 kinase: the N-terminal kinase domain of MAPKAP kinase-1 is essential for peptide IA Leighton, KN Dalby , F Barry Caudwell - FEBS , 1995 - Wiley Online Libraryhttps://febs.onlinelibrary.wiley.com/doi/abs/10.1016/0014-5793(95)01170-J A new peptide conjugate as a highly specific substrate for MAP kinase I Kameshita, A Ishida, H Fujisawa - The journal of biochemistry, 1997 - jstage.jst.go.jphttps://www.jstage.jst.go.jp/article/biochemistry1922/122/1/122_1_168/_article/-char/ja/ In situ fabrication of cleavable peptide arrays on polydimethylsiloxane and applications for kinase activity assays HH Chen, YC Hsiao, JR Li, SH Chen - Analytica Chimica Acta, 2015 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S0003267015000975 A potent synthetic peptide inhibitor of the cAMP-dependent protein kinase. HC Cheng , BE Kemp , RB Pearson , AJ Smith - Journal of Biological , 1986 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S0021925817360416 in a phosphorylation site for cyclic AMP-dependent protein kinase. Comparison of a synthetic peptide with the exposed linking region in the multienzyme polypeptide EA Carrey - Biochemical Journal, 1992 - portlandpress.comhttps://portlandpress.com/biochemj/article-abstract/287/3/791/28764 Irreversible Inactivation of Protein Kinase C Isozymes by Thiol-Reactive Peptide Substrate Analogs CA OBrian, NE Ward, F Chu, JR Stewart - Protein Kinase C Protocols, 2003 - Springerhttps://link.springer.com/protocol/10.1385/1-59259-397-6:441 Mass spectrometric characterization of a series of adenosylated peptides acting as bisubstrate analogs of protein kinases BW Gibson, D Medzihradszky, WM Hines - Journal of the American , 1994 - Springerhttps://link.springer.com/article/10.1016/1044-0305(94)85060-7 Conjugation of monoclonal antibodies to a synthetic peptide substrate for protein kinase: a method for labelling antibodies with 32P BMJ Foxwell, HA Band, J Long, WA Jeffery - British journal of , 1988 - nature.comhttps://www.nature.com/articles/bjc1988112 16 Peptide inhibitors of CAMP-dependent protein kinase BE Kemp , HC Cheng , DA Walsh - Methods in enzymology, 1988 - Elsevierhttps://www.sciencedirect.com/science/article/pii/0076687988590183 Primary Structural Determinants Essential for Potent Inhibition of CAMP-dependent Protein Kinase by Inhibitory Peptides Corresponding to the Active Portion of B David - researchgate.nethttps://www.researchgate.net/profile/Heung-Cheng/publication/20433035_Primary_Structural_Determinants_Essential_for_Potent_Inhibition_of_cAMP-dependent_Protein_Kinase_by_Inhibitory_Peptides_Corresponding_to_the_Active_Portion_of_the_Heat-Stable_Inhibitor_Protein/links/53fdc2370cf2dca800037ca0/Primary-Structural-Determinants-Essential-for-Potent-Inhibition-of-cAMP-dependent-Protein-Kinase-by-Inhibitory-Peptides-Corresponding-to-the-Active-Portion-of-the-Heat-Stable-Inhibitor-Protein.pdf Synthetic design of farnesyl-electrostatic peptides for development of a protein kinase A membrane translocation switch AK Kim, HD Wu , T Inoue - Scientific reports, 2021 - nature.comhttps://www.nature.com/articles/s41598-021-95840-8 reactions with matrix-assisted laser desorption/ionization mass spectrometry and capillary zone electrophoresis: Comparison of the detection efficiency of peptide AG Craig, CA Hoeger, CL Miller - Biological mass , 1994 - Wiley Online Libraryhttps://onlinelibrary.wiley.com/doi/abs/10.1002/bms.1200230810 Steady-state kinetic analysis of protein kinase A interaction with peptide and ATP A Kuznetsov, N Oskolkov , M Hansen - PROCEEDINGS , 2003 - books.google.comhttps://books.google.com/books?hl=en&lr=&id=rtotVS2RKbEC&oi=fnd&pg=PA165&dq=(%22Kemptide%22+OR+%22H-LRRASLG-OH%22)+AND+peptide&ots=Gd10svF6EA&sig=vf_FzVaaMmAneFx7lbWB7TMEYuo Steady-state kinetic analysis of protein kinase A interaction with peptide and ATP A Kuznetsov, N Oskolkov , M Hansen - PROCEEDINGS , 2003 - books.google.comhttps://books.google.com/books?hl=en&lr=&id=rtotVS2RKbEC&oi=fnd&pg=PA165&dq=(%22Kemptide%22+OR+%22H-LRRASLG-OH%22)+AND+peptide&ots=Gd10svF6Hy&sig=Md7UiBCUQ6FzIAqKprcP7ECiXmU Single-subunit allostery in the kinetics of peptide phosphorylation by protein kinase A A Kuznetsov, J Jarv - Proceedings of the Estonian Academy of , 2008 - academia.eduhttps://www.academia.edu/download/52383171/Single-subunit_allostery_in_the_kinetics20170330-24316-1yytpsg.pdf Allosteric cooperativity in inhibition of protein kinase A catalytic subunit A Kuznetsov, J Jarv - Open Enzyme Inhib. J, 2008 - academia.eduhttps://www.academia.edu/download/52383200/Allosteric_Cooperativity_in_Inhibition_o20170330-24322-m7e5a9.pdf Kinetic analysis of inhibition of cAMP-dependent protein kinase catalytic subunit by the peptide-nucleoside conjugate AdcAhxArg6 A Kuznetsov, A Uri , G Raidaru, J Jarv - Bioorganic chemistry, 2004 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S0045206804000343"

 Caspase酶对应的底物，Caspases（半胱氨酸天冬氨酸蛋白酶，半胱氨酸依赖性天冬氨酸定向蛋白酶）是一类蛋白酶家族，其功能与凋亡（程序性细胞死亡），坏死和发烧（炎症）的过程密切相关。

       什么是胱天蛋白酶？

      胱天蛋白酶（Caspases）是含半胱氨酸的天冬氨酸蛋白水解酶，它们是为细胞凋亡的主要介质。多种受体，例如TNF-α 受体，FasL受体，TLR和死亡受体，以及Bcl-2和凋亡抑制剂（IAP）蛋白家族参与并调节该caspase依赖性凋亡途径。一旦Caspase受到上游信号（外部或内在）刺激被激活，即会参与执行下游蛋白底物的水解作用，并触发一系列事件，导致细胞分解，死亡，吞噬作用和细胞碎片的清除。

      人Caspases酶

      人的Caspases家族基于序列相似性和生物学功能等共性主要可分为三大类：第一类由具有长胱天蛋白酶募集结构域的“炎症”胱天蛋白酶组成，他们对P4位上的较大的芳香族或疏水性残基具有亲和力。第二类由具有短的前体结构域的“细胞凋亡效应”胱天蛋白酶组成，而第三类由具有长的前提结构域的Pap位置具有亮氨酸或缬氨酸底物亲和力的“凋亡引发剂”胱天蛋白酶组成（表1）。

       表1. 人胱天蛋白酶的功能分类：

       启动器Caspase和效应器Caspase酶

      根据其在凋亡胱天蛋白酶途径中的作用，胱天蛋白酶可分为两类：启动器和效应器Caspase酶。启动器和效应器Caspas酶都具有由小亚基和大亚基组成的催化位点，Caspase酶的识别位

      凋亡启动器Caspase酶，例如caspase-2，-8，-9和-10可以启动caspase激活级联反应。Caspase-8对于形成死亡诱导信号复合物（DISC）是必不可少的，并且在激活后，Caspase-8激活下游效应子Caspase（例如Caspase 3）并介导线粒体中细胞色素c的释放。Caspase-8已被证明对IETD肽序列具有相对较高的底物选择性。凋亡效应胱天蛋白酶例如Caspase-3，-6和-7虽然不负责启动级联途径，但是当被激活时，它们在级联的中间和后续步骤中起着不可或缺的作用。Caspase-3（CPP32 / apopain）是关键效应器，因为它放大了来自启动器Caspase的信号，使用对Caspase-3有选择性的DEVD肽序列对活化的Caspase-3进行检测，可以检测Caspase-3的活性。

       Caspase酶底物和抑制剂

      Caspase底物和抑制剂由两个关键成分组成：Caspase识别序列和信号产生或蛋白酶抑制基序。不同Caspase识别序列不同，一般由三个或四个氨基酸组成（表2）。Caspase酶识别序列的N端通常有乙酰基（Ac）或碳苯甲氧基（Z）基团修饰，以增强膜的通透性。对应的Caspase识别特定的肽序列为其酶促反应切割位点，释放产生信号或抑制信号的基序。Caspase的显色和荧光底物均以相似的方式起作用，其中底物的信号或颜色强度与蛋白水解活性成正比。

       表2. Caspase的底物及其序列

         Caspase酶的显色底物

      Caspase的显色底物是有Caspase识别序列及生色基团组成，常见的生色团有pNA（对硝基苯胺或4-硝基苯胺），可使用酶标仪或分光光度计在405 nm处进行光密度检测。

       表3. Caspase的显色底物

       Caspase的荧光底物

      Caspase的荧光底物的结构包含与半胱天冬酶识别相关的荧光团，例如7-氨基-4-甲基香豆素（AMC），7-氨基-4-三氟甲基香豆素（AFC）， Rhodamine 110（R110）或ProRed™620。R110的Caspase底物比基于香豆素的Caspase底物（例如AMC和AFC）更敏感，但由于两步裂解过程，其动态范围更窄。 建议将R110标记的Caspase底物用于终点法测定，而将AMC和AFC标记的 Caspase底物用于动力学测定。

      图.从左到右，分别是AMC（7-氨基-4-甲基香豆素），AFC（7-氨基-4-三氟甲基香豆素），Rhodamine 110（R110）和ProRed™620的激发和发射光谱。

       表4.荧光半胱天冬酶底物。

       注意：

        1.ε=在其最大吸收波长处的摩尔消光系数（单位= cm ^-1M ^-1）。

      2.Φ=水性缓冲液（pH 7.2）中的荧光量子产率。

       Caspase抑制剂

      Caspase抑制剂能与Caspase的活性位点结合并形成可逆或不可逆的连接，通常，Caspase抑制剂的结构由Caspase识别序列，诸如醛（-CHO）或氟甲基酮（-FMK）的官能团组成。具有醛官能团的胱天蛋白酶抑制剂是可逆的，而具有FMK的抑制剂是不可逆的。半胱天冬酶底物和抑制剂都具有较小的细胞毒性作用，因此，它们是研究半胱天冬酶活性的有用工具。

       表5. 可逆和不可逆的Caspase酶抑制剂

多肽H2N-Leu-Arg-Arg-Ala-Ser-Leu-Gly-COOH的合成步骤：

1、合成CTC树脂：称取1.0g CTC Resin（如初始取代度约为0.68mmol/g）和0.82mmol Fmoc-Gly-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2.04mmol 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-Gly-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-Gly-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2.04mmol 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4.08mmol DIPEA，1.94mmol 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-Gly-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-Gly-CTC Resin

Fmoc-Ser(tBu)-Leu-Gly-CTC Resin

H2N-Ser(tBu)-Leu-Gly-CTC Resin

Fmoc-Ala-Ser(tBu)-Leu-Gly-CTC Resin

H2N-Ala-Ser(tBu)-Leu-Gly-CTC Resin

Fmoc-Arg(Pbf)-Ala-Ser(tBu)-Leu-Gly-CTC Resin

H2N-Arg(Pbf)-Ala-Ser(tBu)-Leu-Gly-CTC Resin

Fmoc-Arg(Pbf)-Arg(Pbf)-Ala-Ser(tBu)-Leu-Gly-CTC Resin

H2N-Arg(Pbf)-Arg(Pbf)-Ala-Ser(tBu)-Leu-Gly-CTC Resin

Fmoc-Leu-Arg(Pbf)-Arg(Pbf)-Ala-Ser(tBu)-Leu-Gly-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-Leu-Arg(Pbf)-Arg(Pbf)-Ala-Ser(tBu)-Leu-Gly-CTC Resin，结构如下：

5、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H2N-Leu-Arg-Arg-Ala-Ser-Leu-Gly-COOH。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。）

6、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。

7、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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