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Definition

Human chorionic gonadotropin (hCG) is a glycoprotein hormone produced during pregnancy1. It is made by the developing embryo soon after conception1. Its role is to prevent the disintegration of the corpus luteum of the ovary and thereby maintain progesterone production that is critical for a pregnancy in humans1.

Discovery

The activity of hCG was first identified in 1927 when it was demonstrated that the blood and urine of pregnant women contained a gonad-stimulating substance that could induce follicular maturation in immature female mice2.

Classification

hCG is a member of glycoprotein hormone family that also includes LH, FSH and TSH3.

Structural Characteristics

hCG is a heterodimer of two subunits - a and b4.  a-subunit consists of 92 amino acids and two carbohydrate moieties that are linked to Asn52 and Asn784.  The b?-subunit is 145 amino acids long with six carbohydrate moieties attached to Asn13 and Asn304.  It is more acidic that the a- subunit4. 

Mode of action

hCG acts through the LHCG receptor that upon binding of hCG activates the G protein that is bound to the receptor internally that in turn activates cAMP3. cAMP activates the cAMP-dependent protein kinase that in turn triggers the transcription of several target genes in the nucleus3.

Functions

hCG promotes the maintenance of the corpus luteum during the beginning of pregnancy, causing it to secrete the hormone progesterone that enriches the uterus with a thick lining of blood vessels and capillaries so that it can sustain the growing fetus3. Due to its highly-negative charge, hCG may repel the immune cells of the mother, protecting the fetus during the first trimester. It has also been hypothesized that hCG may be a placental link for the development of local maternal immunotolerance5. Because of its similarity to LH, hCG can also be used clinically to induce ovulation in the ovaries as well as testosterone production in the testes3.  It also plays a role in cellular differentiation/proliferation and may activate apoptosis6.  hCG levels are used as a measure during pregnancy testing3.  Human chorionic gonadotropin is extensively used as a parenteral fertility medication3.

References

1.     de Medeiros SF, Norman RJ (2009). Human choriogonadotrophin protein core and sugar branches heterogeneity: basic and clinical insights. Hum Reprod Update, 15(1):69-95.

2.     Ascheim S and Zondek B (1927). Das Hormon des Hypophysenvorderlappens: testobjekt zum nachweis des hormons. Klin Wochenschr 6,248–252.

3.     Stenman UH, Tiitinen A, Alfthan H, Valmu L (2006) The classification, functions and clinical use of different isoforms of HCG. Hum Reprod Update, 12(6):769-84.

4.     Albertini A, Ghielmi S and Belloli S (1982). Structure, immunochemical properties and immunoassay of human chorionic gonadotropin. Intl. J. Clin. & Lab. Res., 12(1), 289-298.

5.     Kayisli U, Selam B, Guzeloglu-Kayisli O, Demir R, Arici A (2003). Human chorionic gonadotropin contributes to maternal immunotolerance and endometrial apoptosis by regulating Fas-Fas ligand system. J. Immunol., 171 (5), 2305–13.

6.     Michels KB, Xue F, Colditz GA, Willett WC (2007). Induced and spontaneous abortion and incidence of breast cancer among young women: a prospective cohort study. Arch. Intern. Med., 167 (8): 814–20.

多肽H2N-Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-COOH的合成步骤：

1、合成CTC树脂：称取1.11g CTC Resin（如初始取代度约为1.18mmol/g）和1.57mmol Fmoc-Gln(Trt)-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3.93mmol 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-Gln(Trt)-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-Gln(Trt)-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3.93mmol Fmoc-Pro-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7.86mmol DIPEA，3.73mmol 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-Pro-Gln(Trt)-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-Pro-Gln(Trt)-CTC Resin

Fmoc-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Ser(tBu)-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Ser(tBu)-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Ser(tBu)-Ser(tBu)-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Ser(tBu)-Ser(tBu)-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Ser(tBu)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Ser(tBu)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Ser(tBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Ser(tBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Asp(OtBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Asp(OtBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Gln(Trt)-Asp(OtBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Gln(Trt)-Asp(OtBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

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

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

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

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

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

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

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

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

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

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

Fmoc-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Pro-Arg(Pbf)-Phe-Gln(Trt)-Asp(OtBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

H2N-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Pro-Arg(Pbf)-Phe-Gln(Trt)-Asp(OtBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-CTC Resin

Fmoc-Thr(tBu)-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Pro-Arg(Pbf)-Phe-Gln(Trt)-Asp(OtBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-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-Thr(tBu)-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Pro-Arg(Pbf)-Phe-Gln(Trt)-Asp(OtBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Ala-Pro-Pro-Pro-Ser(tBu)-Leu-Pro-Ser(tBu)-Pro-Ser(tBu)-Arg(Pbf)-Leu-Pro-Gly-Pro-Ser(tBu)-Asp(OtBu)-Thr(tBu)-Pro-Ile-Leu-Pro-Gln(Trt)-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-Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-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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