HDMNKVLDL.

Definition
Small, anti-inflammatory peptides are useful to inhibit inflammation of a mammal's skin, mucous membranes, or lacerations of the musculature or injury to the brain or leakage of fluids into the air spaces of the lungs.

Discovery
Camussi G in 1986 described platelet-activating factor (PAF) as a phospholipid (1-0-alkyl-2-sn-acetyl-glycero-3-phosphocholine) mediator of inflammation and endotoxic shock. Polymorphonuclear neutrophils (PMN), peritoneal macrophages, vascular endothelial cells, basophils, and platelets synthesize PAF rapidly after appropriate stimuli 1.  For example, TNF or phagocytosis promotes synthesis and release of PAF in PMN or macrophages within 10 min 2.  These stimuli induce phospholipase A2 (PLA2) activity that cleaves membrane phospholipids into lyso-PAF and arachidonic acid. Thrombin stimulates PAFsynthesis in endothelial cells also within 10 min 3.  Other proteinases, such as elastase, stimulate PAF synthesis rapidly in PMN, macrophages, and endothelial cells, and induce PLA2 and acetyltransferase activity. Conversely, different proteinase inhibitors block PAF synthesis induced by TNF. These findings led to the hypothesis that proteinases added to cells and cellular proteinases activated by TNF cleave proteins inhibitory for PLA2, such as lipocortins 4.  Lipocortins belong to a family of related proteins that mediate the anti-inflammatory activity of corticosteroids.

Structural Characteristics
Cloning and sequencing of lipocortins cDNA has provided the amino acid sequence of anti-inflammatory proteins. Steroid induced protein with PLA2 inhibitory activity is uteroglobin, a rabbit secretory protein 5.  Two identical subunits of 70 amino acids form uteroglobin; lipocortin I and II comprise four nonidentical repeats of 70 amino acids. Miele et al., have noticed a striking sequence similarity between amino acid residues 40-46 of uteroglobin and 247-253 of lipocortin I, repeat 3. Synthetic peptides designated "antiflammins" that correspond to such sequences show potent PLA2 inhibitory activity in vitro and an antiinflammatory effect on carrageenan induced rat foot pad edema in vivo 6, 7. SV-IV is a basic, thermostable, secretory protein of low Mr (9758) that is synthesized by rat seminal vesicle (SV) epithelium under strict androgen transcriptional control. By using two different experimental, data were obtained showing that in this protein: (a) the immunomodulatory activity is related to the structural integrity of the whole molecule; (b) the anti-inflammatory activity is located in the N-terminal region of the molecule, the 8-16 peptide fragment being the most active; (c) the identified anti-inflammatory peptide derivatives do not seem to possess pro-coagulant activity, even though this particular function has been located in the 1-70 segment of the molecule 8. The peptide HDMNKVLDL (antiflammin-2) inhibits the synthesis of PAF induced by TNF or phagocytosis in rat macrophages and human neutrophils, and by thrombin in vascular endothelial cells. The peptide MQMKKVLDS (antiflammin-1) is less inhibitory than antiflammin-2 for macrophages and not inhibitory for neutrophils after a 5-min preincubation 9.

Mode of Action
PLA2 are a family of esterases that initiate the arachidonic acid cascade, resulting in the production of numerous inflammatory mediators. Lipocortins are inhibitors of PLA2. Peptides corresponding to a region of high amino-acid sequence similarity between uteroglobin and lipocortin I have potent PLA2 inhibitory activity. Several synthetic peptides corresponding to the region of highest similarity were designed by Miele, L. et al., The most effective anti-inflammatory nonapeptides, termed antiflammins (AFs) correspond to uteroglobin residues 39–47 and lipocortin-1 residues 246–254. Both peptides are PLA2 inhibitors in-vitro and are effective in a classic model of acute inflammation in carrageenan-induced rat footpad edema. The nonapeptides have anti-inflammatory effects in-vitro and in-vivo 10. Antiflammin-1 is inactivated by neutrophils secretory products, possibly oxidizing agents. Synthesis of PAF is inhibited by antiflammin-2 without an appreciable lag, but this inhibition is reversed when neutrophils or macrophages are washed and incubated in fresh medium. Antiflammins must be continuously present to inhibit PAF synthesis. Antiflammins block activation of the acetyltransferase required for PAF synthesis, suggesting that this enzyme is another target for the inhibitory activity of antiflammins. These peptides inhibit neutrophil aggregation and chemotaxis induced by complement component C5a 9.

Functions

Bee venom, in a study of phospholipase-A2,3 nearly 1 kg of freeze-dried bee venom was fractionated and tested all the fractions for anti-inflammatory activity. Experiments showed activity to be associated with the basic peptide fraction 11. 

PAF synthesis, anti-inflammatory peptides inhibit PAF synthesis in PMN, macrophages, and endothelial cells stimulated by TNF, phagocytosis, or proteinases 9.

PMN aggregation and chemotaxis, the antiflammins inhibit also PMN aggregation and chemotaxis, and suppress the inflammatory reaction induced in rat skin by in situ formation of immune complexes or by intradermal injection of TNF and complement component C5a 9.

References

1.     Camussi G (1986). Potential role of platelet-activating factor in renal pathophysiology. Kidney Int., 29: 469-477.
2.      Camussi G, Bussolino F, Salvidio G, Baglioni C (1987). Tumor necrosis factor/cachectin stimulates rat peritoneal macrophages and human endothelial cells to synthesize and release platelet activating factor. J. Exp. Med., 166:1390-1404.
3.     Prescott SM (1984). Human endothelial cells in culture produce platelet-activating factor (1-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine) when stimulated with thrombin. PNAS., 81:3534-3538.
4.     Camussi G, Tetta C, Bussolino F, Baglioni C (1988). Synthesis and release of platelet-activating factor is inhibited by plasma a,proteinase inhibitor or citantichymotrypsin and stimulated by proteinases . J. Exp. Med., 168:1293-1306.
5.     Levin SW, Butler JD, Schumacher UK, Wightman PD, Mukherjee AB (1986). Uteroglobin inhibits phospholipase A2 activity. Life Sci., 38:1813-1819.
6.     Morize I, Surcouf E, Vaney MC, Epelboin Y, Buehner M, Fridlansky F, Milgrom E, Mornon JP (1987) . Refinement of the C222 crystal formofoxidized uteroglobin at 1 .34 A resolution. J. Mol. Biol., 194:725-739.
7.   Miele L, Cordella-Miele E, Facchiano A, Mukherjee AB (1988). Novel anti-inflammatory peptides from the region of highest similarity between uteroglobin and lipocortin I. Nature, 335:726-730.
8.     Ialenti A, Santagada V, Caliendo G, Severino B, Fiorino F, Maffia P, Ianaro A, Morelli F, Di Micco B, Cartenì M, Stiuso P, Metafora V, Metafora S (2001).  Synthesis of novel anti-inflammatory peptides derived from the amino-acid sequence of the bioactive protein SV-IV. FEBS Journal, 268(12):3399-3406.
9.     Camussi G, Tetta C, Bussolino F, Baglioni C (1990). Antiinflamatory peptides (antiinflamins) inhibit synthesis of platelet-activating factor, neutrophil aggregation and chemotaxis, and interdermal inflammatory reactions. J. Exp. Med., 171:913-927.
10.  Sohn J, Kim TI, Yoon YH, Kim JY, Kim SY (2003). Novel transglutaminase inhibitors reverse the inflammation of allergic conjunctivitis. J. Clin. Invest., 111(1):121-128.
11.   Billingham ME, Morley J, Hanson JM, Shipolini RA, Vernon CA (1973). Letter: An anti-inflammatory peptide from bee venom.  Nature, 245(5421):163-164.

多肽H2N-His-Asp-Met-Asn-Lys-Val-Leu-Asp-Leu-COOH的合成步驟：

1、合成CTC樹脂：稱取2.79g CTC Resin（如初始取代度約為0.74mmol/g）和2.48mmol Fmoc-Leu-OH于反應(yīng)器中，加入適量DCM溶解氨基酸（需要注意，此時(shí)CTC樹脂體積會(huì)增大好幾倍，避免DCM溶液過少），再加入6.19mmol DIPEA（Mw：129.1,d：0.740g/ml），反應(yīng)2-3小時(shí)后，可不抽濾溶液，直接加入1ml的HPLC級(jí)甲醇，封端半小時(shí)。依次用DMF洗滌2次，甲醇洗滌1次，DCM洗滌一次，甲醇洗滌一次，DCM洗滌一次，DMF洗滌2次（這里使用甲醇和DCM交替洗滌，是為了更好地去除其他溶質(zhì)，有利于后續(xù)反應(yīng)）。得到  Fmoc-Leu-CTC Resin。結(jié)構(gòu)圖如下：

2、脫Fmoc：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-Leu-CTC 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6.19mmol Fmoc-Asp(OtBu)-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12.39mmol DIPEA，5.88mmol HBTU。反應(yīng)30分鐘后，取小樣洗滌，茚三酮檢測(cè)為無色。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-Asp(OtBu)-Leu-CTC 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-Asp(OtBu)-Leu-CTC Resin

Fmoc-Leu-Asp(OtBu)-Leu-CTC Resin

H2N-Leu-Asp(OtBu)-Leu-CTC Resin

Fmoc-Val-Leu-Asp(OtBu)-Leu-CTC Resin

H2N-Val-Leu-Asp(OtBu)-Leu-CTC Resin

Fmoc-Lys(Boc)-Val-Leu-Asp(OtBu)-Leu-CTC Resin

H2N-Lys(Boc)-Val-Leu-Asp(OtBu)-Leu-CTC Resin

Fmoc-Asn(Trt)-Lys(Boc)-Val-Leu-Asp(OtBu)-Leu-CTC Resin

H2N-Asn(Trt)-Lys(Boc)-Val-Leu-Asp(OtBu)-Leu-CTC Resin

Fmoc-Met-Asn(Trt)-Lys(Boc)-Val-Leu-Asp(OtBu)-Leu-CTC Resin

H2N-Met-Asn(Trt)-Lys(Boc)-Val-Leu-Asp(OtBu)-Leu-CTC Resin

Fmoc-Asp(OtBu)-Met-Asn(Trt)-Lys(Boc)-Val-Leu-Asp(OtBu)-Leu-CTC Resin

H2N-Asp(OtBu)-Met-Asn(Trt)-Lys(Boc)-Val-Leu-Asp(OtBu)-Leu-CTC Resin

Fmoc-His(Trt)-Asp(OtBu)-Met-Asn(Trt)-Lys(Boc)-Val-Leu-Asp(OtBu)-Leu-CTC 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-His(Trt)-Asp(OtBu)-Met-Asn(Trt)-Lys(Boc)-Val-Leu-Asp(OtBu)-Leu-CTC 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，用冰無水乙醚沉淀濾液，并用冰無水乙醚洗滌沉淀物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-His-Asp-Met-Asn-Lys-Val-Leu-Asp-Leu-COOH。結(jié)構(gòu)圖見產(chǎn)品結(jié)構(gòu)圖。

切割液選擇：1）TFA：H2O=95%：5%、TFA：H2O=97.5%：2.5%

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)：

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