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的合成步驟：

1、合成MBHA樹脂：取若干克的MBHA樹脂（如初始取代度為0.5mmol/g）和1倍樹脂摩爾量的Fmoc-Linker-OH加入到反應(yīng)器中，加入DMF，攪拌使氨基酸完全溶解。再加入樹脂2倍量的DIEPA，攪拌混合均勻。再加入樹脂0.95倍量的HBTU，攪拌混合均勻。反應(yīng)3-4小時(shí)后，用DMF洗滌3次。用2倍樹脂體積的10%乙酸酐/DMF 進(jìn)行封端30分鐘。然后再用DMF洗滌3次，甲醇洗滌2次，DCM洗滌2次，再用甲醇洗滌2次。真空干燥12小時(shí)以上，得到干燥的樹脂{Fmoc-Linker-MHBA Resin}，測(cè)定取代度。這里測(cè)得取代度為 0.3mmol/g。結(jié)構(gòu)如下圖：

2、脫Fmoc：取2.45g的上述樹脂，用DCM或DMF溶脹20分鐘。用DMF洗滌2遍。加3倍樹脂體積的20%Pip/DMF溶液，鼓氮?dú)?0分鐘，然后2倍樹脂體積的DMF 洗滌5次。得到 H2N-Linker-MBHA Resin 。（此步驟脫除Fmoc基團(tuán)，茚三酮檢測(cè)為藍(lán)色，Pip為哌啶）。結(jié)構(gòu)圖如下：

3、縮合：取2.21mmol Fmoc-Phe-OH 氨基酸，加入到上述樹脂里，加適當(dāng)DMF溶解氨基酸，再依次加入4.41mmol DIPEA，2.09mmol HBTU。反應(yīng)30分鐘后，取小樣洗滌，茚三酮檢測(cè)為無(wú)色。用2倍樹脂體積的DMF 洗滌3次樹脂。（洗滌樹脂，去掉殘留溶劑，為下一步反應(yīng)做準(zhǔn)備）。得到Fmoc-Phe-Linker-MBHA Resin。氨基酸：DIPEA：HBTU：樹脂=3：6：2.85：1（摩爾比）。結(jié)構(gòu)圖如下：

4、依次循環(huán)步驟二、步驟三，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

以上中間結(jié)構(gòu)，均可在專肽生物多肽計(jì)算器-多肽結(jié)構(gòu)計(jì)算器中，一鍵畫出。

最后再經(jīng)過步驟二得到 H2N-Asn(Trt)-Arg(Pbf)-Asn(Trt)-Phe-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin，結(jié)構(gòu)如下：

5、切割：6倍樹脂體積的切割液（或每1g樹脂加8ml左右的切割液），搖床搖晃 2小時(shí)，過濾掉樹脂，用冰無(wú)水乙醚沉淀濾液，并用冰無(wú)水乙醚洗滌沉淀物3次，最后將沉淀物放真空干燥釜中，常溫干燥24小試，得到粗品H2N-Asn-Arg-Asn-Phe-Leu-Arg-Phe-NH2。結(jié)構(gòu)圖見產(chǎn)品結(jié)構(gòu)圖。

切割液選擇：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%

（前兩種適合沒有容易氧化的氨基酸，例如Trp、Cys、Met。第三種適合幾乎所有的序列。）

6、純化凍干：使用液相色譜純化，收集目標(biāo)峰液體，進(jìn)行凍干，獲得蓬松的粉末狀固體多肽。不過這時(shí)要取小樣復(fù)測(cè)下純度 是否目標(biāo)純度。

7、最后總結(jié)：

杭州專肽生物技術(shù)有限公司（ALLPEPTIDE http://amynixphotography.com）主營(yíng)定制多肽合成業(yè)務(wù)，提供各類長(zhǎng)肽，短肽，環(huán)肽，提供各類修飾肽，如：熒光標(biāo)記修飾（CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等），功能基團(tuán)修飾肽（疊氮、炔基、DBCO、DOTA、NOTA等），同位素標(biāo)記肽（N15、C13），訂書肽（Stapled Peptide）,脂肪酸修飾肽（Pal、Myr、Ste），磷酸化修飾肽（P-Ser、P-Thr、P-Tyr），環(huán)肽（酰胺鍵環(huán)肽、一對(duì)或者多對(duì)二硫鍵環(huán)），生物素標(biāo)記肽，PEG修飾肽，甲基化修飾肽

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