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Definition

Brain natriuretic peptide (BNP) is a 32 amino acid polypeptide that is secreted by the ventricles of the heart in response to stretching of heart muscles1.  

Discovery

BNP was first discovered in porcine brain extracts based on its resemblance to atrial natriuretic peptide (a hormone secreted by mammalian atria) 1.

Classification

BNP belongs to the natriuretic family of peptides that contain three structurally related paracrine factors: Atrial, Brain and C-type natruirectic peptides2. Both atrial and brain natriuretic peptides are secreted in the atria and ventricles of the heart while C-type peptide is secreted in the bone2.  BNP is co-secreted along with a 76 amino acid N-terminal fragment (NT-proBNP) which is biologically inactive3.

Structural Characteristics

BNP is a horseshoe-shaped 32 amino acid peptide that is held by a disulphide bridge formed between amino acids 10 and 264.  It is produced by cleavage of large precursors- prepro and prohormones4. 

Mode of action

BNP activates a transmembrane guanylyl cyclase, natriuretic peptide receptor-A (NPR-A). Activated NPR-A in turn produces the second messenger cGMP that triggers effectors that mediate its cardiac functions5,6.

Functions

BNP similar to ANP decreases systemic vascular resistance and central venous pressure resulting in a decrease in cardiac output and blood volume7.  Both BNP and NT-proBNP levels in the blood are used for screening, diagnosis of acute congestive heart failure (CHF) and may be useful to establish prognosis in heart failure, as both markers are typically higher in patients with worse outcome8. The plasma concentrations of both BNP and NT-proBNP are also typically increased in patients with asymptomatic or symptomatic left ventricular dysfunction8.

BNP plays a very important role in prognostication of millions of diabetics9. It has an improtant role in prognostication of heart surgery patients10. It has been shown that combining BNP with other tools like ICG can improve early diagnosis of heart failure and advance prevention strategies11. BNP can be elevated in renal failure12. The BNP test is used as an aid in the diagnosis and assessment of severity of congestive heart failure (also referred to as heart failure)13.

References

1.     Tetsuji S, Kenji K, Naoto M and Hisayuki M (1988), A new natriuretic peptide in porcine brain, Nature, 332, 78-81.

2.     Potter LR, Yoder AR, Flora DR, Antos LK, Dickey DM (2009), Natruiretic peptides: their structures, receptors, physiologic functions and therapeutic applications, Handb Exp Pharmacol, 191, 341-66.

3.     Bibbins-Domingo K, Gupta R, Na B, Wu AH, Schiller NB, Whooley MA (2007). "N-terminal fragment of the prohormone brain-type natriuretic peptide (NT-proBNP), cardiovascular events, and mortality in patients with stable coronary heart disease". JAMA 297 (2): 169–76.

4.     Brandt I, Lambeir AM, Ketelsleger JM,  Vanderheyden M, Scharpé S and Meest ID, (2006), Dipeptidyl-peptidase IV converts intact B-type natriuretic peptide into its des-SerPro form. Clin. Chem, 52(1): 82-87.

5.     David G. Lowe (1992), Human natriuretic peptide receptor-A guanylyl cyclase is self-associated prior to hormone binding, Biochemistry, 31 (43), pp 10421–10425.

6.     Jun S, Peter M. S and Harvey R. W, (2005),  Differential Effects of cGMP Produced by Soluble and Particulate Guanylyl Cyclase on Mouse Ventricular Myocytes, Experimental Biology and Medicine 230:242-250.

7.     Bhalla V, Willis S, Maisel AS (2004). "B-type natriuretic peptide: the level and the drug--partners in the diagnosis of congestive heart failure". Congest Heart Fail 10(1 Suppl 1): 3–27.

8.     Atisha D, Bhalla MA, Morrison LK, Felicio L, Clopton P, Gardetto N, Kazanegra R, Chiu A, Maisel AS (2004). "A prospective study in search of an optimal B-natriuretic peptide level to screen patients for cardiac dysfunction". Am. Heart J. 148 (3): 518–23.

9.     Bhalla MA, Chiang A, Epshteyn VA, Kazanegra R, Bhalla V, Clopton P, Krishnaswamy P, Morrison LK, Chiu A, Gardetto N, Mudaliar S, Edelman SV, Henry RR, Maisel AS (2004). "Prognostic role of B-type natriuretic peptide levels in patients with type 2 diabetes mellitus". J. Am. Coll. Cardiol. 44 (5): 1047–52.

10.   Lee CY, Burnett JC Jr. (2007), Natruiteric peptides and therapeutic applications, Heart Fail Rev, 12 (2), 131-42

11.   Castellanos LR, Bhalla V, Isakson S, Daniels LB, Bhalla MA, Lin JP, Clopton P, Gardetto N, Hoshino M, Chiu A, Fitzgerald R, Maisel AS (2009). "B-type natriuretic peptide and impedance cardiography at the time of routine echocardiography predict subsequent heart failure events". J. Card. Fail. 15 (1): 41–7.

12.   Bhalla V, Maisel AS (June 2004). "B-type natriuretic peptide. A biomarker for all the right reasons". Ital Heart J 5 (6): 417–20. 

13.   Fitzgerald RL, Cremo R, Gardetto N, Chiu A, Clopton P, Bhalla V, Maisel AS (2005). "Effect of nesiritide in combination with standard therapy on serum concentrations of natriuretic peptides in patients admitted for decompensated congestive heart failure". Am. Heart J. 150 (3): 471–7. 

多肽H2N-Ser-Gln-Asp-Ser-Ala-Phe-Arg-Ile-Gln-Glu-Arg-Leu-Arg-Asn-Ser-Lys-Met-Ala-His-Ser-Ser-Ser-Cys-Phe-Gly-Gln-Lys-Ile-Asp-Arg-Ile-Gly-Ala-Val-Ser-Arg-Leu-Gly-Cys-Asp-Gly-Leu-Arg-Leu-Phe-COOH的合成步骤：

1、合成CTC树脂：称取2.86g CTC Resin（如初始取代度约为0.58mmol/g）和1.99mmol Fmoc-Phe-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4.98mmol 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-Phe-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-Phe-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4.98mmol Fmoc-Leu-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9.95mmol DIPEA，4.73mmol 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-Leu-Phe-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-Leu-Phe-CTC Resin

Fmoc-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Fmoc-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Ile-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Ile-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Arg(Pbf)-Ile-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Arg(Pbf)-Ile-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Phe-Arg(Pbf)-Ile-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Phe-Arg(Pbf)-Ile-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Ala-Phe-Arg(Pbf)-Ile-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Ala-Phe-Arg(Pbf)-Ile-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Ser(tBu)-Ala-Phe-Arg(Pbf)-Ile-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Ser(tBu)-Ala-Phe-Arg(Pbf)-Ile-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Asp(OtBu)-Ser(tBu)-Ala-Phe-Arg(Pbf)-Ile-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Asp(OtBu)-Ser(tBu)-Ala-Phe-Arg(Pbf)-Ile-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Gln(Trt)-Asp(OtBu)-Ser(tBu)-Ala-Phe-Arg(Pbf)-Ile-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

H2N-Gln(Trt)-Asp(OtBu)-Ser(tBu)-Ala-Phe-Arg(Pbf)-Ile-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-CTC Resin

Fmoc-Ser(tBu)-Gln(Trt)-Asp(OtBu)-Ser(tBu)-Ala-Phe-Arg(Pbf)-Ile-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-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-Ser(tBu)-Gln(Trt)-Asp(OtBu)-Ser(tBu)-Ala-Phe-Arg(Pbf)-Ile-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Leu-Arg(Pbf)-Asn(Trt)-Ser(tBu)-Lys(Boc)-Met-Ala-His(Trt)-Ser(tBu)-Ser(tBu)-Ser(tBu)-Cys(Trt)-Phe-Gly-Gln(Trt)-Lys(Boc)-Ile-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Val-Ser(tBu)-Arg(Pbf)-Leu-Gly-Cys(Trt)-Asp(OtBu)-Gly-Leu-Arg(Pbf)-Leu-Phe-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-Ser-Gln-Asp-Ser-Ala-Phe-Arg-Ile-Gln-Glu-Arg-Leu-Arg-Asn-Ser-Lys-Met-Ala-His-Ser-Ser-Ser-Cys-Phe-Gly-Gln-Lys-Ile-Asp-Arg-Ile-Gly-Ala-Val-Ser-Arg-Leu-Gly-Cys-Asp-Gly-Leu-Arg-Leu-Phe-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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