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Preptin is a 34-amino acid peptide hormone, corresponding to Asp⁶⁹-Leu¹⁰² of the proinsulin-like growth factor II E-peptide. It is co-secreted with insulin and amylin from the pancreatic β-cells and is a physiological amplifier of glucose-mediated insulin secretion. It is also anabolic to bone tissue and may contribute to the preservation of bone mass observed in hyperinsulinemic states such as obesity.

Definition
Insulin-like growth factors (IGF)-1 and IGF-2 are ubiquitously expressed peptides with sequence homology to insulin.

Related Peptides
IGFs interacts with a specific receptor on the cell membrane, namely, the IGF-I receptor (IGF-IR), and the interaction is regulated by a group of specific binding proteins. All of these molecules are considered to be members of the IGF family, which includes the polypeptide ligands IGF-I and IGF-II, two types of cell membrane receptors (i.e., IGF-IR and IGF-IIR), and six IGF-binding proteins (i.e., IGFBP-1 through IGFBP-6).

Structural Characteristics
IGFs
IGF-I and IGF-II are single-chain polypeptides. The two molecules have 62% homology in their amino acid sequences. The molecules share additional structural similarities, and their structures resemble the structure of proinsulin1. IGF I consists of 70 amino acid residues, IGF I1 of 67, grouped into domains A and B (similar to insulin), C (analogous to the connecting peptide of proinsulin) and D (not present in insulins). The three intrachain disulfide bridges in IGF 1 and I1 have shown to be located in analogous positions to those in (pro) insulin1.

IGFBPs
The primary structures of mammalian IGFBPs appear to contain three distinct domains of roughly equivalent sizes: the conserved N-terminal domain, the highly variable midregion, and the conserved C-terminal domain.N-terminal domain contains 80–93 amino acid residues after the signal. Ten to 12 of the 16–20 cysteines found in the prepeptides are located within this domain. In IGFBP-1 to -5, these 12 cysteines are fully conserved, whereas in IGFBP-6, 10 of the 12 cysteines are invariant2.Midregion ranging in size from 55 amino acid residues to 95 amino acids separates the N-terminal domain from the C-terminal domain. The amino acid sequence for each midsegment appears to be unique to the protein. C-terminal region are highly conserved and, 6 cysteines of the total 16–20 cysteines are found in the C terminus and are strictly conserved2.

IGF Receptors
Both IGF-IR and IGF-IIR are glycoproteins and are located on the cell membrane. IGF-IR is a tetramer of two identical a-subunits and two identical ß-subunits. Structurally, IGF-IR resembles the insulin receptor, and there is 60% homology between them. IGF-IIR is monomeric. Three ligand-binding regions are found in the extracellular domain of the receptor, one for IGF-II binding and two for proteins containing mannose-6-phosphate (M6P), including renin, proliferin, thyroglobulin, and the latent form of (TGF)-ß transforming growth factor2.

Mode of Action
Binding of IGFs to IGF-IR activates the receptor's tyrosine kinase activity, which triggers a cascade of reactions. Two distinct signal transduction pathways have been identified for IGF-IR. One pathway activates Ras protein, Raf protein, and mitogen-activated protein kinase, and the other pathway involves phosphoinositol-3-kinase. IGF-IR is involved in cell transformation. In vitro experiments have shown that removal of IGF-IR from the cell membrane by eliminating the IGF-IR gene, by suppressing its expression, or by inhibiting its function can abolish cell transformation3. IGFBPs have multiple and complex functions. IGFBPs are able to inhibit or to enhance the action of IGFs, resulting in either suppression or stimulation of cell proliferation. These opposing effects of IGFBPs on IGFs are determined by the molecular structures of the binding proteins. When binding to IGFs, IGFBPs play three major roles: 1) transporting IGFs, 2) protecting IGFs from degradation, and 3) regulating the interaction between IGFs and IGF-IR. Normally, IGFBPs have higher binding affinity to IGFs than does IGF-IR; therefore, binding of IGFBPs to IGFs blocks the interaction between IGFs and IGF-IR and suppresses IGF action. However, binding of IGFBPs to IGFs also protects IGFs from proteolytic degradation, and that protection can enhance the action of IGFs by increasing their bioavailability in local tissue3.

Functions
Direct Involvement in Cancer - IGF-I and IGF-II are strong mitogens for a wide variety of cancer cell lines. Animal experiments indicate that overexpression of IGF-I increase the likelihood of tumor development in certain tissues. The effects of IGFs on cancer cells are mediated through IGF-IR. Eliminating IGF-IR from the cell membrane, blocking the interaction of IGFs with IGF-IR, or interrupting the signal transduction pathway of IGF-IR can abolish the mitogenic action of IGFs on cancer cells. IGF-IR is overexpressed in certain cancers, and its overexpression is associated with aggressive tumors. A recent study indicates that the insulin receptor is involved in mediating the actions of IGF-II on breast cancer. Cancer cells with a strong tendency to metastasize have higher expression of IGF-II and IGF-IR than those with a low ability to do so.In cancer, IGFBPs regulate the action of IGFs. In most situations, the binding proteins suppress the mitogenic action of IGFs and promote apoptosis. It has been shown that IGFBP-3 inhibited breast cancer cell growth without interacting with IGFs4.

IGF I protects and rescues hippocampal neurons against ß-amyloid- and human amylin-induced toxicity - Insulin-like growth factors (IGF-I and IGF-II) are well known trophic factors and their specific receptors are uniquely distributed throughout the brain, being especially concentrated in the hippocampal formation. IGFs possess neurotrophic activities in the hippocampus, an area severely affected in Alzheimer disease. There is evidence that ß-amyloid (aß)-derived peptides likely play an important role in the neurodegenerative process observed in Alzheimer disease, it has been shown that IGFs can be neuroprotective to hippocampal neurons against toxicity induced by amyloidogenic derivatives5.

Reference:

1.     Daughaday WH, Rotwein P (1989). Insulin-like growth factors I and II - Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr. Rev, 10:68–91.

2.     Jones JI, Clemmons DR (1995). Insulin-like growth factors and their binding proteins: biological actions. Endocr. Rev., 16:3–34.

3.     Clemmons DR (1997). Insulin-like growth factor binding proteins and their role in controlling IGF actions. Cytokine Growth Factor Rev, 8:45–62.

4.     Yu H, Rohan T (2000). Role of the Insulin-Like Growth Factor Family in Cancer Development and Progression. Journal of the National Cancer Institute., 92 (18):1472-1489.

5.     Doré S, Kar S, Quirion R (1997). Insulin-like growth factor I protects and rescues hippocampal neurons against ß-amyloid- and human amylin-induced toxicity. Proc. Natl. Acad. Sci, 94:4772–4777.

多肽H2N-Asp-Val-Ser-Thr-Pro-Pro-Thr-Val-Leu-Pro-Asp-Asn-Phe-Pro-Arg-Tyr-Pro-Val-Gly-Lys-Phe-Phe-Gln-Tyr-Asp-Thr-Trp-Lys-Gln-Ser-Thr-Gln-Arg-Leu-COOH的合成步骤：

1、合成CTC树脂：称取2.43g CTC Resin（如初始取代度约为0.73mmol/g）和2.13mmol Fmoc-Leu-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5.32mmol 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-Leu-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-Leu-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5.32mmol Fmoc-Arg(Pbf)-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10.64mmol DIPEA，5.06mmol 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-Arg(Pbf)-Leu-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-Arg(Pbf)-Leu-CTC Resin

Fmoc-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Lys(Boc)-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Lys(Boc)-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Gly-Lys(Boc)-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Gly-Lys(Boc)-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Val-Gly-Lys(Boc)-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Val-Gly-Lys(Boc)-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Pro-Val-Gly-Lys(Boc)-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Pro-Val-Gly-Lys(Boc)-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Tyr(tBu)-Pro-Val-Gly-Lys(Boc)-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Tyr(tBu)-Pro-Val-Gly-Lys(Boc)-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Arg(Pbf)-Tyr(tBu)-Pro-Val-Gly-Lys(Boc)-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Arg(Pbf)-Tyr(tBu)-Pro-Val-Gly-Lys(Boc)-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Pro-Arg(Pbf)-Tyr(tBu)-Pro-Val-Gly-Lys(Boc)-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Pro-Arg(Pbf)-Tyr(tBu)-Pro-Val-Gly-Lys(Boc)-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

Fmoc-Phe-Pro-Arg(Pbf)-Tyr(tBu)-Pro-Val-Gly-Lys(Boc)-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

H2N-Phe-Pro-Arg(Pbf)-Tyr(tBu)-Pro-Val-Gly-Lys(Boc)-Phe-Phe-Gln(Trt)-Tyr(tBu)-Asp(OtBu)-Thr(tBu)-Trp(Boc)-Lys(Boc)-Gln(Trt)-Ser(tBu)-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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