400-998-5282
专注多肽 服务科研
400-998-5282
专注多肽 服务科研
Leptin (22-56), human 是瘦蛋白 (leptin) 的多肽片段，通过 Ob-Rs 的多种亚型起作用。
编号：200184
CAS号：183598-56-3
单字母：H2N-VPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQK-OH
| 参考文献（References）: | Y. Zhang et al., Nature, 372, 425 (1994) M.A. Pelleymounter et al., Science 269, 541 (1995) |
Leptin (22-56), human 是瘦蛋白 (leptin) 的多肽片段，通过 Ob-Rs 的多种亚型起作用。
Leptin (22-56), human is the fragment of leptin, mediated via several isoforms of receptors (Ob-Rs).
瘦素给药产生大鼠抑制食物摄入的片段。
Leptin administration produces fragments of rat inhibition of food intake.
Definition
Leptin is a 16 kDa protein hormone originally discovered in adipose tissue where it acts as a satiety factor in signalling whole body energy balance.
Discovery
In 1950, at Jackson Lab the effects of leptin were observed while studying obese mice with random mutation 1. The obese yellow mice were attaining weights up to 75 or 80 grams. Later leptin was discovered in 1994 by Jeffrey M. Friedman et al., through the study of such mice. Mutation of ob (obese gene) resulted in profound obesity and type II diabetes as part of a syndrome that resembles morbid obesity in humans 2.
Structural Characteristics
Leptin of humans has 146 amino acid sequence containing one disulphide bond. Its molecular weight is around 16 kDa. Leptin has 67% sequence identity among diverse species. Leptin is a four-helix bundle with one very short strand segment and two relatively long interconnected loops. This is consistent with a classification as a cytokine four-helix bundle 3.
Mode of Action
Leptin acts via specific receptors (Ob-R), of which six isoforms are at present recognized (from Ob-Ra to Ob-Rf). Ob-Rb is the only isoform able to activate JAK-STAT and MAPK signaling cascades. A large body of evidence suggests that leptin and its receptors are involved in prostate physiology and pathophysiology in humans. Ob-R in rat is involved in the autocrine-paracrine functional regulation of the epithelial cells of adult rat seminal vesicles and prostate 4. Once leptin has bound to the Ob-Rb receptor, it activates the stat3, which is phosphorylated and travels to the nucleus to, presumably, effect changes in gene expression. One of the main effects on gene expression is the down-regulation of the expression of endocannabinoids, responsible for increasing appetite. In response to leptin, receptor neurons have been shown to remodel themselves, changing the number and types of synapses that fire onto them. Mutation of ob results in profound obesity and type II diabetes as part of a syndrome that resembles morbid obesity in humans. The ob gene product may function as part of a signalling pathway from adipose tissue that acts to regulate the size of the body fat depot 5.
Functions
Adiposity signal, Leptin is an adiposity hormone that modulates the activity of multiple hypothalamic signaling pathways involved in the control of food intake. Administration of leptin or one of its downstream mediators, neuropeptide Y (NPY), could affect food intake by modulating the brain stem neurophysiological response to ascending meal-related feedback signals in the nucleus of the solitary tract (NTS) in anesthetized male Long-Evans rats 6.
Appetite control, leptin binds to NPY neurons in the arculate nucleus, in such a way that decreases the activity of these neurons. Leptin signals to the brain that the body has had enough to eat, or satiety. A very small group of humans possess homozygous mutations for the leptin gene which leads to a constant desire for food, resulting in severe obesity.
Modulation of T cell activity, the role of Leptin/Leptin receptors in modulation of T cell activity in immune system was shown in experimentation with mice. Leptin/leptin receptor pathway is involved in the modulation of the regulatory immune response in atherosclerosis, and alteration in regulatory immunity may predispose obese individuals to atherosclerosis 7.
Lung surfactant activity, in fetal lung leptin is induced in the alveolar interstitial fibroblasts ("lipofibroblasts") by the action of PTHrP secreted by formative alveolar epithelium (endoderm) under moderate stretch. The leptin from the mesenchyme in turn acts back on the epithelium at the leptin receptor carried in the alveolar type II pneumocytes and induces surfactant expression which is one of the main functions of these type II pneumocytes 8.
Reproduction, it appears that leptin and estradiol interact coordinately in a concentration-dependent manner to control IVF outcome. In mice, leptin is also required for male and female fertility. In mammals, ovulatory cycles in females are linked to energy balance (positive or negative depending on whether a female is losing or gaining weight) and energy flux (how much energy is consumed and expended) much more than energy status (fat levels) 9.
References
1. Ingalls AM, Dickie MM, Snell GD (1950). Obese, a new mutation in the house mouse. J. Hered., 41(12):317-318.
2. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM (1994). Positional cloning of the mouse obese gene and its human homologue. Nature, 372(6505):425–432.
3. Zhang F, Basinski MB, Beals JM, Briggs SL, Churgay LM, Clawson DK, DiMarchi RD, Furman TC, Hale JE, Hsiung HM, Schoner BE, Smith DP, Zhang XY, Wery JP, Schevitz RW (1997). Crystal structure of the obese protein leptin-E100. Nature, 387:206-209..
4. Malendowicz W, Rucinski M, Macchi C, Spinazzi R, Ziolkowska A, Nussdorfer GG, Kwias Z (2006). Leptin and leptin receptors in the prostate and seminal vesicles of the adult rat. Int. J. Mol. Med., 18(4):615–618.
5. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM (1994). Positional cloning of the mouse obese gene and its human homologue. Nature, 372(6505):425–432.
6. Williams KW, Scott MM, Elmquist JK (2009). From observation to experimentation: leptin action in the mediobasal hypothalamus. Am. J. Clin. Nutr., 89(3):985–990.
7. Taleb S, Herbin O, Ait-Oufella H, Verreth W, Gourdy P, Barateau V, Merval R, Esposito B, Clément K, Holvoet P, Tedgui A, Mallat Z (2007). Defective leptin/leptin receptor signaling improves regulatory T cell immune response and protects mice from atherosclerosis.. Arterioscler Thromb Vasc Biol., 27(12):2691–2698.
8. Torday JS, Rehan VK (2006). Up-regulation of fetal rat lung parathyroid hormone-related protein gene regulatory network down-regulates the Sonic Hedgehog/Wnt/betacatenin gene regulatory network. Pediatr Res., 60(4):382–388.
9. Anifandis G, Koutselini E, Louridas K, Liakopoulos V, Leivaditis K, Mantzavinos T, Sioutopoulou D, Vamvakopoulos N (2005). Estradiol and leptin as conditional prognostic IVF markers. Reproduction, 129(4):531–534.
| DOI | 名称 | |
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| 10.1111/j.1365-2265.2004.01999.x | Elevated leptin fragments in renal failure correlate with BMI and haematopoiesis and are normalized by haemodialysis | 下载 |
多肽H2N-Val-Pro-Ile-Gln-Lys-Val-Gln-Asp-Asp-Thr-Lys-Thr-Leu-Ile-Lys-Thr-Ile-Val-Thr-Arg-Ile-Asn-Asp-Ile-Ser-His-Thr-Gln-Ser-Val-Ser-Ser-Lys-Gln-Lys-COOH的合成步骤：
1、合成CTC树脂：称取1.82g CTC Resin（如初始取代度约为0.43mmol/g）和0.94mmol Fmoc-Lys(Boc)-OH于反应器中，加入适量DCM溶解氨基酸（需要注意，此时CTC树脂体积会增大好几倍，避免DCM溶液过少），再加入2.35mmol DIPEA（Mw：129.1,d：0.740g/ml），反应2-3小时后，可不抽滤溶液，直接加入1ml的HPLC级甲醇，封端半小时。依次用DMF洗涤2次，甲醇洗涤1次，DCM洗涤一次，甲醇洗涤一次，DCM洗涤一次，DMF洗涤2次（这里使用甲醇和DCM交替洗涤，是为了更好地去除其他溶质，有利于后续反应）。得到 Fmoc-Lys(Boc)-CTC Resin。结构图如下：
2、脱Fmoc：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-Lys(Boc)-CTC 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2.35mmol Fmoc-Gln(Trt)-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4.7mmol DIPEA，2.23mmol 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-Gln(Trt)-Lys(Boc)-CTC 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-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Asp(OtBu)-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Gln(Trt)-Asp(OtBu)-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Gln(Trt)-Asp(OtBu)-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Val-Gln(Trt)-Asp(OtBu)-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Val-Gln(Trt)-Asp(OtBu)-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Lys(Boc)-Val-Gln(Trt)-Asp(OtBu)-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Lys(Boc)-Val-Gln(Trt)-Asp(OtBu)-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Gln(Trt)-Lys(Boc)-Val-Gln(Trt)-Asp(OtBu)-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Gln(Trt)-Lys(Boc)-Val-Gln(Trt)-Asp(OtBu)-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Ile-Gln(Trt)-Lys(Boc)-Val-Gln(Trt)-Asp(OtBu)-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Ile-Gln(Trt)-Lys(Boc)-Val-Gln(Trt)-Asp(OtBu)-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Pro-Ile-Gln(Trt)-Lys(Boc)-Val-Gln(Trt)-Asp(OtBu)-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
H2N-Pro-Ile-Gln(Trt)-Lys(Boc)-Val-Gln(Trt)-Asp(OtBu)-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC Resin
Fmoc-Val-Pro-Ile-Gln(Trt)-Lys(Boc)-Val-Gln(Trt)-Asp(OtBu)-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC 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-Val-Pro-Ile-Gln(Trt)-Lys(Boc)-Val-Gln(Trt)-Asp(OtBu)-Asp(OtBu)-Thr(tBu)-Lys(Boc)-Thr(tBu)-Leu-Ile-Lys(Boc)-Thr(tBu)-Ile-Val-Thr(tBu)-Arg(Pbf)-Ile-Asn(Trt)-Asp(OtBu)-Ile-Ser(tBu)-His(Trt)-Thr(tBu)-Gln(Trt)-Ser(tBu)-Val-Ser(tBu)-Ser(tBu)-Lys(Boc)-Gln(Trt)-Lys(Boc)-CTC 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-Val-Pro-Ile-Gln-Lys-Val-Gln-Asp-Asp-Thr-Lys-Thr-Leu-Ile-Lys-Thr-Ile-Val-Thr-Arg-Ile-Asn-Asp-Ile-Ser-His-Thr-Gln-Ser-Val-Ser-Ser-Lys-Gln-Lys-COOH。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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