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（D-精氨酸，D-苯丙氨酸⁵,D-Trp⁷·⁹,Leu´´）-最初被描述为低效力胃饥饿素受体拮抗剂的物质P出人意料地被发现是一种完全反向激动剂（EC₅₀ = 5.2 nM）。在用胃促生长素受体瞬时转染的COS-7细胞中，物质P类似物rPKPfQwFwLL将组成型信号传导降低到未转染细胞中观察到的水平。假设胃促生长素受体的组成型信号传导在食欲控制的调节中具有生理相关性，胃促生长素接收器的反向激动剂可能对治疗肥胖感兴趣。因此，Asakawa等人发现，外周给药H-6395降低了瘦小鼠、饮食诱导肥胖小鼠和肥胖小鼠的食物摄入量。

(D-Arg¹,D-Phe⁵,D-Trp⁷·⁹,Leu¹¹)-Substance P initially described as a low potency ghrelin receptor antagonist has surprisingly been found to be a full inverse agonist (EC₅₀ = 5.2 nM). In COS-7 cells transiently transfected with the ghrelin receptor the substance P analog rPKPfQwFwLL decreased the constitutive signaling down to levels observed in untransfected cells. Assuming that constitutive signaling of the ghrelin receptor is of physiological relevance in the regulation of appetite control, inverse agonists of the ghrelin receptor could be interesting for the treatment of obesity. So, Asakawa et al. found that peripherally administered H-6395 decreased food intake in lean mice, in mice with diet induced obesity, and in ob/ob obese mice.

[D-Arg1，D-Phe5，D-Trp7,9，Leu11]-物质P醋酸盐是一种广谱的神经肽反向激动剂和拮抗剂。它作为胃促生长素受体的完全反向激动剂（EC50=5.2 nM）和速激肽、缓激肽、CCK和蛙皮素受体的拮抗剂。在体外诱导癌症细胞凋亡并抑制其生长。

[D-Arg1,D-Phe5,D-Trp7,9,Leu11]-Substance P acetate is a broad-spectrum neuropeptide inverse agonist and antagonist. It acts as a full inverse agonist for the ghrelin receptor (EC50 = 5.2 nM) and antagonist at tachykinin, bradykinin, CCK, and bombesin receptors. It induces apoptosis and inhibits cancer cell growth in vitro.

Substance P is a neuropeptide that belongs to the Feeding Regulatory Peptides family. It is synthesized in the hypothalamus and secreted by neurons in the spinal cord, brainstem, and gut. Substance P has been shown to regulate feeding behavior through its interaction with opioid receptors and the release of other neurotransmitters. The synthetic form of this peptide has been shown to reduce body fat in animal models and increased sensitivity to insulin in diabetic patients. This peptide also reduces camp levels in rats, which may be due to its ability to stimulate secretion of camp from pancreatic cells.

P物质（Substance P）的定义

P物质（SP）是一种十一肽，在外周和中枢神经系统中都很丰富，它通常与经典的神经递质之一（最常见的是血清素（5-HT））共定位【1】。

Substance P (SP) an undecapeptide, is abundant both in the periphery and in the CNS, where it is usually co-localised with one of the classical neurotransmitters, most commonly serotonin (5- HT) 【1】.

P物质（Substance P）的相关肽

SP属于一种神经肽家族，称为速激肽，具有共同的C端序列：Phe-X-Gly-Leu-Met-NH2。三种最常见的速激肽是SP，神经激肽A（NKA）和神经激肽B（NKB）；它们的生物学作用是通过指定为NK1，NK2和NK3的特定细胞表面受体介导的，SP是NK1受体的首选激动剂，NK2受体的NKA和NK3受体的NKB【2】。

SP belongs to a family of neuropeptides known as tachykinins that share the common C-terminal sequence: Phe-X-Gly-Leu-Met-NH2. The three most common tachykinins are SP, neurokinin A (NKA), and neurokinin B (NKB); their biologic actions are mediated through specific cell-surface receptors designated NK1, NK2, and NK3, with SP the preferred agonist for NK1 receptors, NKA for NK2 receptors, and NKB for NK3 receptors 【2】.

P物质（Substance P）的发现

SP最初是由von Euler和Gaddum于1931年发现的，是一种在体外引起肠道收缩的组织提取物；在随后的几十年中，进一步研究了其生物学作用和组织分布【3】。

SP was originally discovered in 1931 by von Euler and Gaddum as a tissue extract that caused intestinal contraction in vitro; its biologic actions and tissue distribution were further investigated over subsequent decades 【3】.

P物质（Substance P）的结构特征

SP是一种11个残基的神经肽，序列为Arg-Pro-Lys-Pro-Gln-Glin-Phe-Gly-Leu-Met-NH2）【4】。在一项研究中，将SP的C端和N端片段与亲本分子进行比较，以了解它们的能力：（a）收缩分离的豚鼠回肠，（b）诱导大鼠流涎，（C）激发单个猫背角神经元，以及（d）通过小鼠颅内注射诱导抓挠。与七肽一样小的C末端片段在所有测定系统上都是有效的SP激动剂。含有五个氨基酸或更少的C末端片段最多只有弱活性。N末端片段在分离的豚鼠回肠上完全无活性。然而，在大鼠唾液和中枢神经系统测定中，N末端片段能够产生弱的SP样活性【5】。获得的结果表明，虽然SP的羧基末端对于肽的支气管活性至关重要，但氨基末端肽（多达四个残基）的丢失实际上增强了支气管收缩剂对肽的反应。这种增强的部分原因似乎是SP和SP5-11的酶促降解差异所致。数据表明，二肽基氨肽酶切割SP可以增强其生物活性【6】。SP类似物：Senktide（琥珀酰-[Asp6，Me-Phe8]SP-（6-11））是NK-3（SP-N）受体的选择性类似物，其效力是SP的20-100倍，比驻留在肌肉细胞上的NK-1（SP-P）受体的选择性类似物强约1000倍【7】。在小鼠鞘内注射后，研究了五种SP类似物对神经激肽（NK）1受体激动剂如SP，physalaemin和（p-Glu6，Pro9）-SP（6-11）（septide）诱导的舔，咬和抓挠反应的影响。肽产生类似SP的行为反应，其效力约为D-Pro9类似物D-七肽的25倍。较低剂量的（D-Arg1，D-Pro2,4，D-Phe7，D-His9，Leu11）-SP比（D-Phe7，D-His9，Leu11）-SP（6-11）显着降低了Septide诱导的反应。相反，（D-Arg1，D-Pro2,4，D-Phe7，D-His9）-SP（0.5-1.0 nmol）和（D-Phe7，D-His9）-SP（6-11）（0.5-2.0 nmol）仅抑制SP诱导的行为反应，而不抑制藻毒素或七肽诱导的反应。这项研究的结果表明，NK-1受体激动剂不一定受到含有D-His 的SP类似物的相同程度的影响【8】。P物质[D-Arg1，D-Phe5，D-Trp7,9，Leu11]SP（SpD）和[Arg6，D-Trp7,9，NmePhe8]P物质的类似物可抑制神经肽刺激的Ca2+动员，酪氨酸磷酸化和ERK激活。至关重要的是，SpD和[Arg6，D-Trp7,9，NmePhe8]SP在体内和体外抑制SCLC细胞生长并刺激SCLC细胞凋亡。SP类似物最初被表征为“广谱神经肽拮抗剂”【9】。

SP is an 11-residue neuropeptide with the sequence Arg-Pro-Lys-Pro-Gln-Glin-Phe-Gly-Leu-Met-NH2) 【4】. In a study, the C- and N-terminal fragments of SP were compared to the parent molecule with respect to their ability to: (a) contract the isolated guinea pig ileum, (b) induce salivation in the rat, (c) excite single cat dorsal horn neurones, and (d) induce scratching by intracranial injections in mice. C-terminal fragments as small as the heptapeptide were potent SP agonists on all assay systems. C-terminal fragments containing five amino acids or less were, at most, only weakly active. N-terminal fragments were totally inactive on the isolated guinea pig ileum. On the rat salivation and central nervous system assays, however, N-terminal fragments were capable of weak SP-like activity 【5】. The results obtained, indicated that while the carboxy terminal of SP is essential for peptide bronchoactivity, loss of amino terminal peptides (up to four residues) actually enhances bronchoconstrictor responses to the peptide. Part of this enhancement appears to result from differences in the enzymatic degradation of SP and SP5-11. The data suggest that cleavage of SP by dipeptidyl aminopeptidases could enhance its bioactivity 【6】. SP analogs: Senktide (succinyl-[Asp6,Me-Phe8]SP-(6-11)), a selective analog for the NK-3 (SP-N) receptor, is 20-100 times more potent than SP and about 1000-fold more potent than the selective analogs for the NK-1 (SP-P) receptor, which resides on muscle cells 【7】. Effects of five SP analogs on the licking, biting and scratching response induced by neurokinin (NK) 1 receptor agonists such as SP, physalaemin and (p-Glu6,Pro9)-SP (6-11) (septide) were studied after intrathecal injections in mice. Peptide brought about a SP-like behavioral response, and was approximately 25 times more potent than the D-Pro9 analog, D-septide. Septide-induced response was significantly reduced by lower doses of (D-Arg1, D-Pro2,4, D-Phe7, D-His9, Leu11)-SP than (D- Phe7, D-His9, Leu11)-SP (6-11). In contrast, (D-Arg1, D-Pro2,4, D-Phe7, D-His9)-SP (0.5-1.0 nmol) and (D-Phe7, D-His9)-SP (6-11) (0.5-2.0 nmol) inhibited only SP-induced behavioral response, but not physalaemin- or septide-induced response. The results of this study indicate that NK-1 receptor agonists are not necessarily affected to a same degree by SP analogs containing D-His 8. Analogues of substance P, [D-Arg1,D-Phe5,D-Trp7,9,Leu11] SP (SpD) and [Arg6,D-Trp7,9,NmePhe8]substance P can inhibit neuropeptide-stimulated Ca2+ mobilization, tyrosine phosphorylation, and ERK activation . Crucially, SpD and [Arg6,D-Trp7,9,NmePhe8] SP inhibit SCLC cell growth in vivo and in vitro and stimulate SCLC cell apoptosis. SP analogues were characterized originally as "broad spectrum neuropeptide antagonists" 【9】.

P物质（Substance P）的作用方式

SP受体是一种G蛋白偶联受体，在许多方面类似于精神病学中其他经过充分研究的受体，特别是单胺受体2。SP与其受体的相互作用激活了Gq，Gq又激活了磷脂酶C，将磷脂酰肌醇二磷酸分解为肌醇三磷酸（IP3）和二酰基甘油（DAG）。IP3作用于肌浆网中的特定受体以释放细胞内的Ca2+，而DAG通过蛋白激酶C作用于打开质膜中的L型钙通道。细胞内[Ca2+]的升高诱导组织反应。与SP所见的一系列不同的行动，有许多治疗可能性【10】。

The SP receptor is a G protein-coupled receptor, in many respects similar to other well-studied receptors in psychiatry, particularly monoamine receptors 2.  The interaction of SP with its receptor activates Gq, which in turn activates phospholipase C to break down phosphatidyl inositol bisphosphate into inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 acts on specific receptors in the sarcoplasmic reticulum to release intracellular stores of Ca2+, while DAG acts via protein kinase C to open L-type calcium channels in the plasma membrane. The rise in intracellular [Ca2+] induces the tissue response. With an array of actions as diverse as that seen with SP, there is scope for numerous therapeutic possibilities 【10】.

P物质（Substance P）的功能

在中枢神经系统中，SP与情绪障碍，焦虑，压力，强化，神经发生，神经毒性和疼痛的调节有关。在消化道中，SP和其他一些速激肽是调节运动活动，离子和液体分泌以及血管功能的神经递质【11，12】。

In the central nervous system, SP is associated with the regulation of mood disorders, anxiety, stress, reinforcement, neurogenesis, neurotoxicity and pain.  In the digestive tract, SP, along with some other tachykinins, are neurotransmitters that regulate motor activity, secretion of ions and fluid, as well as vascular functions 【11，12】.

P物质（Substance P）的相关文献

1. Argyropoulos SV, Nutt DJ (2000). Substance P antagonists: novel agents in the treatment of depression. Expert Opin Investig Drugs,  9(8):1871-1875.
2. Book: Substance P and Related Tachykinins. Chapter 13: Neuropsychopharmacology: By Nadia MJ, Kramer MS.
3. Senba E, Tohyama M (1985). Origin and fine structure of substance P-containing nerve terminals in the facial nucleus of the rat:an immunohistochemical study. Exp Brain Res., 57(3):537-546.
4. Seidel MF, Tsalik J, Vetter H, Müller W (2007). Substance P in Rheumatic Diseases. Current Rheumatology Reviews, 3:17-30.
5. Piercey MF, Dobry PJ, Einspahr FJ, Schroeder LA, Masiques N (1982) Use of substance P fragments to differentiate substance P receptors of different tissues. Regulatory Peptides, 3(5-6):337-349.
6. Shore SA, Drazen JM (1988). Airway responses to substance P and substance P fragments in the guinea pig. Pulm Pharmacol., 1(3):113-118.
7. Hanani M, Chorev M, Gilon C, Selinger Z (1988). The actions of receptor-selective substance P analogs on myenteric neurons: an electrophysiological investigation. European journal of pharmacology, 153(2-3):247-253.
8. Sakurada T, Yamada T, Tan-no K, Manome Y, Sakurada S, Kisara K, Ohba M (1991). Differential effects of substance P analogs on neurokinin 1 receptor agonists in the mouse spinal cord. J Pharmacol Exp Ther.,  259:205-210
9. MacKinnon AC, Waters C, Jodrell D, Haslett C, Sethi T (2001). Bombesin and Substance P Analogues Differentially Regulate G-protein Coupling to the Bombesin Receptor. J. Biol. Chem.,   276(30):28083-28091..
10. Khawaja AM, Rogers DF (1996). Tachykinins: receptor to effector. Int J Biochem Cell Biol.,  28(7):721-738.
11.  Leeman SE, Mroz EA (1974).  Substance P. Life Sci., 15(12):2033–2044.
12. Wiesenfeld-Hallin Z, Xu XJ (1993). The differential roles of substance P and neurokinin A in spinal cord hyperexcitability and neurogenic inflammation. Regul Pept., 46(1-2):165-173

很多蛋白在细胞中非常容易被降解，或被标记，进而被选择性地破坏。但含有部分D型氨基酸的多肽则显示了很强的抵抗蛋白酶降解能力。

多肽H2N-DArg-Pro-Lys-Pro-DPhe-Gln-DTrp-Phe-DTrp-Leu-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1.55g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1.4mmol 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2.79mmol DIPEA，1.33mmol 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-Leu-Leu-Linker-MBHA Resin

H2N-Leu-Leu-Linker-MBHA Resin

Fmoc-DTrp(Boc)-Leu-Leu-Linker-MBHA Resin

H2N-DTrp(Boc)-Leu-Leu-Linker-MBHA Resin

Fmoc-Phe-DTrp(Boc)-Leu-Leu-Linker-MBHA Resin

H2N-Phe-DTrp(Boc)-Leu-Leu-Linker-MBHA Resin

Fmoc-DTrp(Boc)-Phe-DTrp(Boc)-Leu-Leu-Linker-MBHA Resin

H2N-DTrp(Boc)-Phe-DTrp(Boc)-Leu-Leu-Linker-MBHA Resin

Fmoc-Gln(Trt)-DTrp(Boc)-Phe-DTrp(Boc)-Leu-Leu-Linker-MBHA Resin

H2N-Gln(Trt)-DTrp(Boc)-Phe-DTrp(Boc)-Leu-Leu-Linker-MBHA Resin

Fmoc-DPhe-Gln(Trt)-DTrp(Boc)-Phe-DTrp(Boc)-Leu-Leu-Linker-MBHA Resin

H2N-DPhe-Gln(Trt)-DTrp(Boc)-Phe-DTrp(Boc)-Leu-Leu-Linker-MBHA Resin

Fmoc-Pro-DPhe-Gln(Trt)-DTrp(Boc)-Phe-DTrp(Boc)-Leu-Leu-Linker-MBHA Resin

H2N-Pro-DPhe-Gln(Trt)-DTrp(Boc)-Phe-DTrp(Boc)-Leu-Leu-Linker-MBHA Resin

Fmoc-Lys(Boc)-Pro-DPhe-Gln(Trt)-DTrp(Boc)-Phe-DTrp(Boc)-Leu-Leu-Linker-MBHA Resin

H2N-Lys(Boc)-Pro-DPhe-Gln(Trt)-DTrp(Boc)-Phe-DTrp(Boc)-Leu-Leu-Linker-MBHA Resin

Fmoc-Pro-Lys(Boc)-Pro-DPhe-Gln(Trt)-DTrp(Boc)-Phe-DTrp(Boc)-Leu-Leu-Linker-MBHA Resin

H2N-Pro-Lys(Boc)-Pro-DPhe-Gln(Trt)-DTrp(Boc)-Phe-DTrp(Boc)-Leu-Leu-Linker-MBHA Resin

Fmoc-DArg(Pbf)-Pro-Lys(Boc)-Pro-DPhe-Gln(Trt)-DTrp(Boc)-Phe-DTrp(Boc)-Leu-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-DArg(Pbf)-Pro-Lys(Boc)-Pro-DPhe-Gln(Trt)-DTrp(Boc)-Phe-DTrp(Boc)-Leu-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-DArg-Pro-Lys-Pro-DPhe-Gln-DTrp-Phe-DTrp-Leu-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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