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Kisspeptin-54(human) TFA (Metastin(human) TFA) 是一种 Kisspeptin 受体 (KISS1，GPR54) 的内源性配体，以高亲和力结合大鼠和人类 GPR54 受体，其 Ki 值分别为 1.81 nM 和 1.45 nM。Kisspeptin-54(human) TFA 可抑制肿瘤转移并刺激促性腺激素分泌。

Kisspeptin-54(human) TFA (Metastin(human) TFA) is an endogenous ligand for kisspeptin receptor (KISS1, GPR54). Kisspeptin-54(human) TFA binds to rat and human GPR54 receptors with Ki values of 1.81 nM and 1.45 nM, respectively. Kisspeptin-54(human) TFA hinders tumor metastasis and stimulates gonadotropin secretion.

Metastin（人）是G蛋白偶联孤儿受体如OT7T175（也称为AXOR12）和GPR54的内源性配体。Kisspeptin-54最初被称为“metastin”，因为它具有抑制肿瘤转移的能力。该肽被认为是人类KISS1基因的主要产物。它以高亲和力与大鼠和人KISS1受体结合，Ki值分别为1.80和1.45nM。

Metastin (human) is an endogenous ligand to G-protein-coupled orphan receptors such as OT7T175 (also known as AXOR12) and GPR54. Kisspeptin-54 was initially termed as "metastin" because of its capacity to inhibit tumor metastasis. This peptide has been considered as the major product of the human KISS1 gene. It binds with high affinity to rat and human KISS1 receptors with Ki values of 1.80 and 1.45 nM respectively.

Definition
The KISS1 receptor (previously designated GPR54) has been paired with biologically active cleavage peptides of the KiSS-1 gene product, the kisspeptins (KP).

Discovery
KP was originally identified in 1996 from a metastasis suppressor gene, KiSS-1, in malignant melanomas. Initially, the largest cleavage product, KP-54, was identified for its ability to suppress metastatic potential in human melanoma cells. Its expression also resulted in suppression of melanoma metastasis in athymic nude mice and it was therefore termed metastin1.  In 2001, one of the orphans KISS1 (previously designated GPR54, AXOR12, hOT7T175) was paired with three biologically active cleavage peptides of the kisspeptin gene (KiSS-1) gene product isolated from human placenta, the KP’s  (KP)-54, KP-13 and KP-10 2, 3. The KP, as a collective group, where individual KP is referred to, their amino acid sequence length e.g., KP-54, KP-13 and KP-10.

Structural Characteristics
The full-length KP protein (KP-145) has a PEST sequence (proline, glutamic acid, serine, threonine and aspartic acid residue-rich sequence). This motif predisposes proteins for ubiquitination and proteosome degradation and suggests that cytosolic KP-145 would have a short half-life 4.  In rat and mouse, the longest KP cleavage fragment is composed of 52 amino acids. Although overall homology of human KiSS-1 gene products to rat and mouse is relatively low (B52%), KP-10 is highly conserved between human, mouse and rat, with only one amino acid difference in the sequence between species 1.  

Mode of Action
Activation of KISS1 (GPR54) results in intracellular calcium mobilization that is not affected by pertussis toxin and does not result in changes in cAMP accumulation, suggesting that it is a Gq-coupled receptor. KP activation of KISS1 (GPR54) has been shown to simultaneously result in release of arachidonic acid and stimulation of the mitogen-activated protein kinase (MAPKs) extracellular signal-regulated kinase (ERK) 1 and ERK2. This has been attributed to increased phosphorylation of MAPK 5

Functions

KP and matrix metalloproteinases- Downregulation of one or both of the gelatinase matrix metalloproteinases (MMPs), MMP-2 and MMP-9, by KP has been shown. KP has been described as regulator of MMPs at both the transcriptional and protein level. Transcriptional changes have been shown not to function through the MAPK signalling pathways. Instead, nuclear factor-kB binding to the MMP-9 promoter region, necessary for expression of MMP-9 6.

KP and cancer metastasis- Direct interaction of KiSS-1 has been identified with two transcription factors, activator protein-2a and specificity protein-1, both of which have been shown to be important regulators of genes involved in tumourigenesis, metastasis and development 7.

KP and placentation- Microarray analysis confirmed the higher KISS1 (GPR54) expression in first trimester invasive trophoblasts, when compared to non/low-invasive term cells.

KISS1 (GPR54) – an unexpected molecular switch for puberty- In 2003, three different groups identified KISS1 (GPR54) as an unexpected molecular switch for puberty 8.

KP and GnRH release-The signalling pathway showed that injection of KP-10 and KP-54 directly into the lateral cerebral ventricle of the mouse brain potently stimulated LH and follicle-stimulating hormone (FSH) secretion, an effect that could be blocked by pretreatment with the GnRH antagonist acyline 8.

KP neurones in the brain- Approximately 75% of GnRH neurones co-express KISS1 (GPR54), a finding that has been confirmed in sheep, where intra cerebral injection of KP resulted in direct release of GnRH into the cerebrospinal fluid 8.

Direct effects of KP on the testes-Continuous chronic administration of KP in male rats resulted in decreased testicular weight and degeneration of the seminiferous tubules, leading to the hypothesis that KP may alter testicular blood flow 8.

References

1.     Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE, Welch DR. (1996). KiSS-1, a novel human malignant melanoma metastasissuppressor gene. J Natl Cancer Inst., 88: 1731-1737.

2.     Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul E, Brézillon S, Tyldesley R, Suarez-Huerta N, Vandeput F, Blanpain C, Schiffmann SN, Vassart G, Parmentier M. (2001). The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol Chem., 276(37):34631-34636.

3.     Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, Terao Y, Kumano S, Takatsu Y, Masuda Y, Ishibashi Y, Watanabe T, Asada M, Yamada T, Suenaga M, Kitada C, Usuki S, Kurokawa T, Onda H, Nishimura O, Fujino M. (2001). Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature., 411 (6837): 613–617.

4.     Harms JF, Welch DR, Miele ME (2003). KISS1 metastasis suppression and emergent pathways. Clin Exp Metastasis., 20(1):11-18.

5.     Becker JA, Mirjolet JF, Bernard J, Burgeon E, Simons MJ, Vassart G, Parmentier M, Libert F. (2005). Activation of GPR54 promotes cell cycle arrest and apoptosis of human tumor cells through a specific transcriptional program not shared by other Gq-coupled receptors. Biochem Biophys Res Commun., 326(3):677-686.

6.     Qiao C, Wang CH, Shang T, Lin QD (2005). Clinical significance of KiSS-1 and matrix metalloproteinase-9 expression in trophoblasts of women with preeclampsia and their relation to perinatal outcome of neonates. Zhonghua Fu Chan Ke Za Zhi., 40(9): 585–590.

7.     Mitchell DC, Abdelrahim M, Weng J, Stafford LJ, Safe S, Bar-Eli M, Liu M.(2006). Regulation of KiSS-1 metastasis suppressor gene expression in breast cancer cells by direct interaction of transcription factors activator protein-2alpha and specificity protein-1. J Biol Chem., 281: 51–58.

8.     Mead EJ, Maguire JJ, Kuc RE,Davenport AP (2007). Kisspeptins: a multifunctional peptide system with a role in reproduction, cancer and the cardiovascular system. Br J Pharmacol., 151 (8):1143-1153.

M L Gottsch, et al. A Role for Kisspeptins in the Regulation of Gonadotropin Secretion in the Mouse. Endocrinology. 2004 Sep;145(9):4073-7. : https://pubmed.ncbi.nlm.nih.gov/15217982
M Kotani, et al. The Metastasis Suppressor Gene KiSS-1 Encodes Kisspeptins, the Natural Ligands of the Orphan G Protein-Coupled Receptor GPR54. J Biol Chem. 2001 Sep 14;276(37):34631-6. : https://pubmed.ncbi.nlm.nih.gov/11457843

多肽H2N-Gly-Thr-Ser-Leu-Ser-Pro-Pro-Pro-Glu-Ser-Ser-Gly-Ser-Arg-Gln-Gln-Pro-Gly-Leu-Ser-Ala-Pro-His-Ser-Arg-Gln-Ile-Pro-Ala-Pro-Gln-Gly-Ala-Val-Leu-Val-Gln-Arg-Glu-Lys-Asp-Leu-Pro-Asn-Tyr-Asn-Trp-Asn-Ser-Phe-Gly-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1.47g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1.32mmol 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2.65mmol DIPEA，1.26mmol 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-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

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

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

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

Fmoc-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Fmoc-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Fmoc-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Pro-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Pro-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Pro-Pro-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Pro-Pro-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Pro-Pro-Pro-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Pro-Pro-Pro-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Ser(tBu)-Pro-Pro-Pro-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Ser(tBu)-Pro-Pro-Pro-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Leu-Ser(tBu)-Pro-Pro-Pro-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Leu-Ser(tBu)-Pro-Pro-Pro-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Ser(tBu)-Leu-Ser(tBu)-Pro-Pro-Pro-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Ser(tBu)-Leu-Ser(tBu)-Pro-Pro-Pro-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Thr(tBu)-Ser(tBu)-Leu-Ser(tBu)-Pro-Pro-Pro-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

H2N-Thr(tBu)-Ser(tBu)-Leu-Ser(tBu)-Pro-Pro-Pro-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-Leu-Arg(Pbf)-Phe-Linker-MBHA Resin

Fmoc-Gly-Thr(tBu)-Ser(tBu)-Leu-Ser(tBu)-Pro-Pro-Pro-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-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-Gly-Thr(tBu)-Ser(tBu)-Leu-Ser(tBu)-Pro-Pro-Pro-Glu(OtBu)-Ser(tBu)-Ser(tBu)-Gly-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Gln(Trt)-Pro-Gly-Leu-Ser(tBu)-Ala-Pro-His(Trt)-Ser(tBu)-Arg(Pbf)-Gln(Trt)-Ile-Pro-Ala-Pro-Gln(Trt)-Gly-Ala-Val-Leu-Val-Gln(Trt)-Arg(Pbf)-Glu(OtBu)-Lys(Boc)-Asp(OtBu)-Leu-Pro-Asn(Trt)-Tyr(tBu)-Asn(Trt)-Trp(Boc)-Asn(Trt)-Ser(tBu)-Phe-Gly-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-Gly-Thr-Ser-Leu-Ser-Pro-Pro-Pro-Glu-Ser-Ser-Gly-Ser-Arg-Gln-Gln-Pro-Gly-Leu-Ser-Ala-Pro-His-Ser-Arg-Gln-Ile-Pro-Ala-Pro-Gln-Gly-Ala-Val-Leu-Val-Gln-Arg-Glu-Lys-Asp-Leu-Pro-Asn-Tyr-Asn-Trp-Asn-Ser-Phe-Gly-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，甲基化修饰肽等。

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