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Autocamtide 2, amide是钙调蛋白激酶 (CaMK) 家族实验的底物(100 μM final concentration)。
Autocamtide 2, amide is a substrate (100 μM final concentration) for CaMK family assays.

Definition
Protein kinases are transferase that catalyze the phosphorylation of proteins by covalently attaching phosphate groups to them, using ATP as a phosphate donor. Reversible protein phosphorylation-dephosphorylation has a principal role in the regulation of essentially all cellular functions. Kinase/phosphatase substrates can be found grouped according to their kinase families. A single substrate can have a many number of modifications.

Discovery
Phoebus AL at the Rockefeller Institute identified phosphate in the protein Vitellin (phosvitin) in 1906 1 and by 1933 Fritz Lipmann had detected phosphoserine in Casein 2. In 1954 Eugene P. Kennedy described the first ‘enzymatic phosphorylation of proteins’ in a variety of normal and malignant tissues, he showed that the phosphorus of the phosphoprotein fraction undergoes a high rate of turnover, as measured by incorporation of 32P 3 .

Structural Characteristics
There are thousands of different kinds of proteins in any particular cell that are substrate for different kinases and phosphatases. Phosphorylation of any site on a given protein can change the structure, function or localization of that protein. Within a protein, phosphorylation can occur on several amino acids. Phosphorylation on serine is the most common, followed by threonine. Tyrosine phosphorylation is relatively rare. However, since tyrosine phosphorylated proteins are relatively easy to purify using antibodies, tyrosine phosphorylation sites are relatively well understood. Histidine and aspartate phosphorylation occurs in prokaryotes as part of two-component signaling and in some cases in eukaryotes in some signal transduction pathways 4. Phosphorylation of seryl or threonyl (and occasionally tyrosyl) residues triggers small conformational changes in these proteins that alter their biological properties.

Mode of Action
Phosphatase removes a phosphate group from its substrate by hydrolysing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group. Protein kinases are the effectors of phosphorylation and catalyse the transfer of a y-phosphate from ATP to specific amino acids on proteins. The addition of a phosphate (PO4) molecule to a polar R group of an amino acid residue can turn a hydrophobic portion of a protein into a polar and extremely hydrophilic portion of molecule. In this way it can introduce a conformational change in the structure of the protein via interaction with other hydrophobic and hydrophilic residues in the protein. Several protein kinases are important in cellular control (e.g. glycogen synthase kinase-3, acetyl CoA carboxylase kinase, tyrosine hydroxylase kinase and casein kinase-2), which are themselves controlled by allosteric effectors, phosphorylation, insulin and other growth factors, or by regulators. Protein phosphatase catalytic units are responsible for dephosphorylating many regulated proteins in the cytoplasm that are phosphorylated on serine and threonine residues. Some protein phosphatases are controlled by second messengers. PP-1(Protein phosphatase-1) is regulated by cyclic AMP in several ways that vary with the form of the enzyme and the tissue. It is inhibited by cyclic AMP through the phosphorylation of inhibitor-1 and its isoforms  through the phosphorylation of targeting proteins such as the glycogen-binding subunit, and through allosteric inhibition by phosphorylase a. PP-2B (Protein phosphatase-2B)  is activated by Ca2+ through the interaction of this second messenger with an integral Ca2+ -binding subunit, as well as calmodulin itself. Protein phosphorylation-dephosphorylation is the basis of a network of interlocking systems that allow hormones and other extracellular signals, acting through just a few second messengers, to coordinate biochemical functions 5.

Functions
Reversible phosphorylation of proteins is an important regulatory mechanism that occurs in living cells 6. Reversible phosphorylation results in a change in conformation the structure in many enzymes and receptors, causing them to become activated or deactivated.
Regulatory roles, the p53 protein is heavily regulated through phosphorylation sites, it has 18 different phosphorylation sites. Activation and phosphorylation of p53 can lead to cell cycle arrest, which can be reversed under some circumstances, or apoptotic cell death 7. In energy-requiring reactions, phosphorylation of Na+/K+-ATPase during the transport of sodium (Na+) and potassium(K+) ions across the cell membrane in osmoregulation to maintain homeostasis.
Enzyme regulation, phosphorylation of the enzyme GSK-3 by AKT (Protein kinase B) is a important regulation in insulin signaling pathway.
Protein-protein interaction, phosphorylation of the cytosolic components of NADPH oxidase, a large membrane-bound, multi-protein enzyme plays an important role in the regulation of protein-protein interactions of the enzyme.
Protein degradation, phosphorylation of some proteins causes them to be degraded by the ATP-dependent ubiquitin/proteasome pathway. These proteins become substrates for particular E3 ubiquitin ligases only when they are phosphorylated 8.

References

1.     Levene PA, Alsberg CL (1906). The cleavage products of vitellin. J. Biol. Chem., 2(1): 127-133.

2.     Lipmann FA, Levene PA (1932). Serinephosphoric acid obtained on hydrolysis of vitellinic acid. J. Biol. Chem., 98 (1):109-114.

3.     Burnett G, Kennedy EP (1954). The enzymatic phosphorylation of proteins. J. Biol. Chem., 211(2):969–980.

4.     Aumailley M, Bruckner-Tuderman L, Carter WG, Deutzmann R, Edgar D, Ekblom P, Engel J, Engvall E, Hohenester E, Jones JC, Kleinman HK, Marinkovich MP, Martin GR, Mayer U, Meneguzzi G, Miner JH, Miyazaki K, Patarroyo M, Paulsson M, Quaranta V, Sanes JR, Sasaki T, Sekiguchi K, Sorokin LM, Talts JF, Tryggvason K, Uitto J, Virtanen I, von der Mark K, Wewer UM, Yamada Y, Yurchenco PD (2005). A simplified laminin nomenclature. Matrix Biol., 24(5):326-332.

5.     Cohen P (1988). Protein Phosphorylation and Hormone Action. Proceedings of the Royal Society of London. Biological Sciences, 234(1275):115-144.

6.     Barford D, Das AK, Egloff MP (1998). The structure and mechanism of protein phosphatases: insights into catalysis and regulation. Annu Rev Biophys Biomol Struct., 27:133–164.

7.     Ashcroft M, Kubbutat MH, Vousden KH (1999). Regulation of p53 function and stability by phosphorylation. Mol. Cell. Biol., 19(3):1751–1758.

8.     Babior BM (1999). NADPH oxidase: an update. Blood, 93(5):1464–1476.

多肽H2N-Lys-Lys-Ala-Leu-Arg-Arg-Gln-Glu-Thr-Val-Asp-Ala-Leu-NH2的合成步骤：

1、合成MBHA树脂：取若干克的MBHA树脂（如初始取代度为0.5mmol/g）和1倍树脂摩尔量的Fmoc-Linker-OH加入到反应器中，加入DMF，搅拌使氨基酸完全溶解。再加入树脂2倍量的DIEPA，搅拌混合均匀。再加入树脂0.95倍量的HBTU，搅拌混合均匀。反应3-4小时后，用DMF洗涤3次。用2倍树脂体积的10%乙酸酐/DMF 进行封端30分钟。然后再用DMF洗涤3次，甲醇洗涤2次，DCM洗涤2次，再用甲醇洗涤2次。真空干燥12小时以上，得到干燥的树脂{Fmoc-Linker-MHBA Resin}，测定取代度。这里测得取代度为 0.3mmol/g。结构如下图：

2、脱Fmoc：取2.5g的上述树脂，用DCM或DMF溶胀20分钟。用DMF洗涤2遍。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-Linker-MBHA 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2.25mmol 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4.5mmol DIPEA，2.14mmol 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-Linker-MBHA 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-Linker-MBHA Resin

Fmoc-Ala-Leu-Linker-MBHA Resin

H2N-Ala-Leu-Linker-MBHA Resin

Fmoc-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

H2N-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

Fmoc-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

H2N-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

Fmoc-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

H2N-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

H2N-Glu(OtBu)-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

Fmoc-Gln(Trt)-Glu(OtBu)-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

H2N-Gln(Trt)-Glu(OtBu)-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Gln(Trt)-Glu(OtBu)-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

H2N-Arg(Pbf)-Gln(Trt)-Glu(OtBu)-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Glu(OtBu)-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

H2N-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Glu(OtBu)-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

Fmoc-Leu-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Glu(OtBu)-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

H2N-Leu-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Glu(OtBu)-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

Fmoc-Ala-Leu-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Glu(OtBu)-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

H2N-Ala-Leu-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Glu(OtBu)-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

Fmoc-Lys(Boc)-Ala-Leu-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Glu(OtBu)-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

H2N-Lys(Boc)-Ala-Leu-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Glu(OtBu)-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA Resin

Fmoc-Lys(Boc)-Lys(Boc)-Ala-Leu-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Glu(OtBu)-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA 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-Lys(Boc)-Lys(Boc)-Ala-Leu-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Glu(OtBu)-Thr(tBu)-Val-Asp(OtBu)-Ala-Leu-Linker-MBHA 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-Lys-Lys-Ala-Leu-Arg-Arg-Gln-Glu-Thr-Val-Asp-Ala-Leu-NH2。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。）

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，甲基化修饰肽等。

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