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

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.

多肽H2N-Asn-Arg-Asn-Phe-Leu-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2.45g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2.21mmol 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4.41mmol DIPEA，2.09mmol 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-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

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

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

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

Fmoc-Asn(Trt)-Phe-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Asn(Trt)-Phe-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Asn(Trt)-Phe-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Arg(Pbf)-Asn(Trt)-Phe-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Asn(Trt)-Arg(Pbf)-Asn(Trt)-Phe-Leu-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-Asn(Trt)-Arg(Pbf)-Asn(Trt)-Phe-Leu-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-Asn-Arg-Asn-Phe-Leu-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

以上所有内容，为专肽生物原创内容，请勿发布到其他网站上。