专肽生物_定制多肽合成服务_多肽合成定制服务_多肽合成厂家_多肽合成公司

Neuropeptide AF (human) 是内源性的抗阿片 (opioid) 肽。
Neuropeptide AF (human) is an endogenous antiopioid peptide.

定义
神经肽的长度为3-40个氨基酸，可作为神经递质。它们广泛分布于中枢神经系统和周围神经系统。

发现
神经肽是由约翰·休斯博士和科斯特里茨博士于1975年发现的。它们是内啡肽，内在产生的吗啡样物质，会在体内产生一系列类似药物的作用。可以从序列信息1中鉴定神经肽前体mRNA序列，并且得到的翻译蛋白序列包括信号肽序列和一个或多个神经肽。广泛而复杂的一系列酶处理步骤，包括被激素或前蛋白转化酶切割以及其他翻译后修饰，在创建活性神经肽之前就发生在翻译后的蛋白质序列上  2,3。

结构特征
通过核磁共振（NMR）光谱研究了几种来自软体动物的类似神经肽的构象性质。肽的N末端可变区中的氨基酸取代对溶液中反向转化的种群具有显着影响。通过使用两个独立的NMR参数测得的转弯数，发现使用Helix aspersa的受体膜制剂与IC50值高度相关（r2 = 0.93和0.82）。这些结果表明，构象集合降低了特定肽相对于特定受体4,5的有效浓度。

神经肽Y与人肽相同，并且与禽胰多肽高度同源。神经肽Y和禽胰多肽之间的同源性保留了维持三级结构必不可少的所有残基。结果表明，神经肽保留了紧凑的三级结构，其特征是在N末端的聚脯氨酸II类螺旋和C末端的a螺旋 6之间广泛的疏水相互作用。

已经通过许多孤儿受体之一发现了一些肽，这些受体是内源性配体未知的受体，例如“类阿片受体样1”（ORL1）。随后，已阐明该ORL1受体的内源性激动剂的结构，一种称为孤儿蛋白FQ或伤害感受蛋白的17个氨基酸的肽7。

行动方式
神经肽是由神经元作为细胞间信使释放的肽。一些神经肽充当神经递质，而另一些充当激素。神经肽既可以为我们提供支持，也可以为我们提供帮助。抗炎神经肽可帮助我们减少皮肤发炎。神经肽是自然产生的，可以在非常有限的时间内与靶细胞膜受体在明确的作用位点相互作用。因此，大多数这些内源性化合物的特征在于低的生物屏障渗透性和非常高的酶促降解敏感性。脑室内或全身注射神经肽Y（NPY）可使cast割的雌性大鼠血浆中的促黄体生成激素（LH）水平降低。6。

功能

生物功能，神经肽控制着我们的情绪，能量水平，痛苦和愉悦感，体重以及解决问题的能力；它们还会形成记忆，情感行为，食欲和发炎，修复疤痕和皱纹并调节我们的免疫系统。这些活跃的大脑小信使实际上打开了皮肤7的细胞功能。因此，今天，与神经肽系统相互作用的药物设计是后基因组药物化学研究最广泛的途径之一。

P物质已被确定为负责伤害性信号传递的主要神经肽。内源性阿片类药物是天然神经肽，负责伤害性信号的调节（通常是抑制）。

免疫系统，当它们被分泌时，它们会激活自然杀伤细胞（NK细胞），从而增强我们的免疫系统。

随着内啡肽的分泌越来越多，血管病变使收缩的血管恢复到正常状态，使血液以正常方式流动。大多数成人疾病都始于血管堵塞。内啡肽有助于改善血液循环。

内啡肽通过去除超氧化物具有抗衰老作用。从呼吸进入人体的氧气可以转变为超氧化物。这是造成人类疾病和衰老的最大敌人之一。

抗压力激素，应对压力的能力与我们体内的内啡肽水平成正比。

缓解疼痛的作用是，我们的神经系统在接收到疼痛信号时会分泌神经递质。一旦内啡肽在疼痛的那一刻被释放，内啡肽就会与神经元上的内啡肽受体结合，从而阻止第一种神经递质被分泌出来。

记忆力，神经肽可以改善记忆力，因为它们可以使脑细胞保持年轻健康。

参考

1.     Hummon AB, Richmond TA, Verleyen P, Baggerman G, Huybrechts J, Ewing MA, Vierstraete E, Rodriguez-Zas SL, Liliane SL, Robinson GE (2006). From the genome to the proteome: uncovering peptides in the Apis brain. Science, 27(314):647-649.

2.     Rockwell NC, Krysan DJ, Komiyama T, Fuller RS (2002). Precursor processing by Kex2/Furin Proteases. Chem. Rev., 102:4525–4548.

3.     Von ER, Beck-Sickinger AG (2004). Biosynthesis of peptide hormones derived from precursor sequences. Curr. Med. Chem.,11:2651–2665.

4.     Edison AS, Espinoza E, Zachariah C (1999). Conformational Ensembles: The Role of Neuropeptide Structures in Receptor Binding. The Journal of Neuroscience., 19(15):6318-6326.

5.     Payza K, Greenberg MJ, Price DA (1989). Further characterization of Helix FMRFamide receptors: kinetics, tissue distribution, and interactions with the endogenous heptapeptides. Peptides, 10:657-661.

6.     Allen J, Novotný J, Martin J, Heinrich G (1987). Molecular structure of mammalian neuropeptide Y: Analysis by molecular cloning and computer-aided comparison with crystal structure of avian homologue. PNAS., 84:2532-2536.

7.     Guya J, Lia S,  Pelletier G (1988). Studies on the physiological role and mechanism of action of neuropeptide Y in the regulation of luteinizing hormone secretion in the rat. Regulatory Peptides., 23(2):209-216.

Definition
Neuropeptides with the Arg-Phe-amide motif at their C termini (RFamide peptides) were identified in the brains of several vertebrates, and shown to have important physiological roles in neuroendocrine, behavioral, sensory, and autonomic functions.

Discovery
Price DA, Greenberg MJ in 1977 studied the structure of a molluscan cardioexcitatory neuropeptide. Neuropeptides with Arg-Phe-amide (RFamide) motif at their C termini, which were found in the ganglia of the venus clam, (FMRFamide), have been identified in the brains of several vertebrates and referred to as RFamide peptide(s). A chicken pentapeptide (LPLRFPamide) has also been isolated from its brain. Two pain modulatory neuropeptides [FF and AF], prolactin-releasing peptide (PrRP), gonadotropin- inhibitory hormone,and GH-releasing peptide are also RFamide peptides. To date, these RFamide-peptides have had important physiological roles in neuroendocrine, behavioral, sensory, and autonomic functions. Two PrRPs consisting of 31 amino acids (PrRP31) and 20 amino acids (PrRP20) from bovine hypothalamus extract were potent stimulators of prolactin (PRL) release as an endogenous ligand of an orphan G protein-coupled receptor (hGR3). Immunocytochemical studies showed that, in rat, PrRP cell bodies were located in the brain and hypothalamus, and that their nerve fibers projected into a wide range of areas in the brain 1,2,3.

Structural Characteristics
FMRF-amide-related peptides (FaRPs) are small peptides of 4–18 amino acids with RFamide (arg-phe-NH2) at the C terminus. Neuropeptides with the Arg-Phe-amide motif at their C termini (RFamide peptides) were identified in the brains of several vertebrates. Moriyama S et al., (2007) identified RFamide peptides, which are teleost prolactin-releasing peptide (PrRP) homologs, in the sea lamprey, Petromyzon marinus and characterized their effect on the release of pituitary hormones in vitro. Two RFamide peptides (RFa-A and RFa-B) were isolated from an acid extract of sea lamprey brain, including hypothalamus by Sep-Pak C18 cartridge, affinity chromatography using anti-salmon PrRP serum, and reverse-phase HPLC on an ODS-120T column. Amino acid sequences and mass spectrometric analyses revealed that RFa-A and RFa-B consist of 25  and 20 aa, respectively, and have 75% sequence identity within the C-terminal 20 aa. The RFa-B cDNA encoding a preprohormone of 142 aa was cloned from the lamprey brain, and the deduced aa sequence from positions 48–67 was identical to the sequence of RFa-B  4. 

Mode of Action
The potency (muscle force-generated) of a number of long-chain RFamide neuropeptides has been examined. Many of the heptapeptides, octapeptides and the decapeptide LMS were found to induce greater contraction than FMRFamide in both smooth muscles and in both species. RFamide neuropeptides interacted with the neurotransmitter acetylcholine in an additive way and RFamide-induced contractions were inhibited by the neuromodulator serotonin. Pre-treatment with a calcium-free saline completely abolished acetylcholine-induced responses but only partially inhibited RFamide responses in the muscles, suggesting that acetylcholine acts to cause influx of extracellular calcium for contraction. Result suggests that an additional involvement of a fast calcium channel is present in the RFamide responses. Force regulation in these muscles appears to result from a complex interaction of RFamide neuropeptides with the primary transmitter acetylcholine and the neuromodulator serotonin 5.

Unlike in mammals, a few RFamide peptide fibers were projected to the pituitary, and terminated close to PRL producing cells in the rostral pars distalis (RPD) and to the somatolactin somatolactin (SL)-producing cells in the pars intermedia (PI) in rainbow trout. On the basis of the localization of salmon RFamide peptide, compared its hypophysiotropic effects on the release of three evolutionarily related hormones, PRL and SL. Salmon RFamide peptide stimulated PRL release from the pituitary both in vivo and in vitro, as well as in tilapia. Salmon RFamide peptide also affected SL releases from the pituitary  6,7.

Functions

Regulation of PRL release, these results indicate that RFamide peptide is a major hypothalamic peptide involved in the regulation of PRL release and that this peptide may exist throughout vertebrate evolution 6.

RFamide during the development of a primary polyp, antisera to the sequence Arg-Phe-amide (RF-amide) have a high affinity to the nervous system of fixed hydroid polyps. Incubation of Hydractinia echinata gastrozooids with RFamide antisera visualizes an extremely dense plexus of neuronal processes in body and head regions. A ring of sensory cells around the mouth opening is the first group of neurons to show RFamide immunoreactivity during the development of a primary polyp 8.

Two RFamide peptides in lamprey were identified, which are structurally related to teleost PrRP, by peptide isolation and cDNA cloning from lamprey brain/hypothalamus. Evidence suggests that RFamide peptides are major hypothalamic and/or pituitary peptides that may be involved in inhibition of GH and MSH release in lamprey 4.

References

1.     Price DA, Greenberg MJ (1977). Structure of a molluscan cardioexcitatory neuropeptide. Science, 197:670–671.

2.      Dockray GJ, Reeve Jr JR, Shively J, Gayton RJ, Barnard CS (1983). A novel active pentapeptide from chicken brain identified by antibodies to FMRFamide. Nature, 305:328-330.

3.     Yang HY, Fratta W, Majane EA, Costa E (1985). Isolation, sequencing, synthesis, and pharmacological characterization of two brain neuropeptides that modulate the action of morphine. PNAS., 82:7757-77614.

 4.    Moriyama S, Kasahara M, Amiya N, Takahashi A, Amano M, Sower SA, Yamamori K, Kawauchi H (2007). RFamide peptides inhibit the expression of melanotropin and growth hormone genes in the pituitary of an Agnathan, the sea lamprey, Petromyzon marinus. Endocrinology, 148(8):3740-3749.
5.     Moulis A, Huddart H (2004). RFamide neuropeptide actions on molluscan proboscis smooth muscle: interactions with primary neurotransmitters J Comp Physiol B., 174(5):363-370.

6.     Moriyama S, Ito T, Takahashi A, Amano M, Sower SA, Hirano T, Yamamori K, Kawauchi H (2002). A homolog of mammalian PRL-releasing peptide (fish arginyl-phenylalanyl-amide peptide) is a major hypothalamic peptide of PRL release in teleost fish. Endocrinology, 143:2071-2079.

7.     Sakamoto T, Agustsson T, Moriyama S, Itoh T, Takahashi A, Kawauchi H, Björnsson BT, Ando M (2003). Intra-arterial injection of prolactin-releasing peptide elevates prolactin gene expression and plasma prolactin levels in rainbow trout. J Comp Physiol., 173:333-337.

8.     Grimmelikhuijzen CJP (1985). Antisera to the sequence Arg-Phe-amide visualize neuronal centralization in hydroid polyps. Cell and Tissue Research., 241(1):171-182.

Payne ME, et al. Calcium/calmodulin-dependent protein kinase II. Characterization of distinct calmodulin binding and inhibitory domains. J Biol Chem. 1988 May 25;263(15):7190-5. : https://www.ncbi.nlm.nih.gov/pubmed/2835367

多肽H2N-Ala-Gly-Glu-Gly-Leu-Asn-Ser-Gln-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-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1.29g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1.16mmol Fmoc-Phe-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2.32mmol DIPEA，1.1mmol 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-Phe-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-Phe-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Gln(Trt)-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Gln(Trt)-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Ser(tBu)-Gln(Trt)-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Ser(tBu)-Gln(Trt)-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Asn(Trt)-Ser(tBu)-Gln(Trt)-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Asn(Trt)-Ser(tBu)-Gln(Trt)-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Leu-Asn(Trt)-Ser(tBu)-Gln(Trt)-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Leu-Asn(Trt)-Ser(tBu)-Gln(Trt)-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Gly-Leu-Asn(Trt)-Ser(tBu)-Gln(Trt)-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Gly-Leu-Asn(Trt)-Ser(tBu)-Gln(Trt)-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Gly-Leu-Asn(Trt)-Ser(tBu)-Gln(Trt)-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Glu(OtBu)-Gly-Leu-Asn(Trt)-Ser(tBu)-Gln(Trt)-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Gly-Glu(OtBu)-Gly-Leu-Asn(Trt)-Ser(tBu)-Gln(Trt)-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Gly-Glu(OtBu)-Gly-Leu-Asn(Trt)-Ser(tBu)-Gln(Trt)-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Ala-Gly-Glu(OtBu)-Gly-Leu-Asn(Trt)-Ser(tBu)-Gln(Trt)-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-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-Ala-Gly-Glu(OtBu)-Gly-Leu-Asn(Trt)-Ser(tBu)-Gln(Trt)-Phe-Trp(Boc)-Ser(tBu)-Leu-Ala-Ala-Pro-Gln(Trt)-Arg(Pbf)-Phe-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-Ala-Gly-Glu-Gly-Leu-Asn-Ser-Gln-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-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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