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Definition

Charybdotoxin (CTX) is a 37 amino acid neurotoxin from the venom of the scorpion Leiurus quinquestriatus hebraeus that blocks calcium-activated potassium channels that causes hyperexcitation of the nervous system1.

Discovery

CTX was first identified and partially purified from the venom of the scorpion, Leiurus quinquestriatus hebraeus in 19852.  It was found that CTX could reversibly inhibit Ca2+-activated K+ channels of mammalian skeletal muscle2.

Classification

CTX belongs to the charybdotoxin family of potassion channel blocking peptides3.

Structural Characteristics

CTX like other potassium channel blocking peptides has a characteristic fold in its peptide backbone3. It also has three disulphide bridges that hold the three stranded beta-sheet to the two to three turn alpha-helix in place3. The toxicity of CTX depends on the Lys at position 273.  Several structural analogs of CTX have been designed4.

Mode of action

CTX blocks potassium channels by binding to a receptor at the opening of the ion conduction pathway5.  It binds both to the open and the closed states5. In addition, the block is enhanced as the ionic strength is lowered6.

Functions

Scorpions such as the deathstalker paralyse their prey by injecting a potent mix of peptide toxins one of which is CTX7. Anti-scorpion venom serum (AScVS) is an effective and safe method of therapy in severe scorpion envenoming syndrome. Compared with other therapies like alpha blockers it has a relatively short recovery period8.  CTX is also a good model to study potassium channels7.

References

1.     Laurent F, Michel A, Bonnet PA, Chapat JP, Boucard M (1993). Evaluation of the relaxant effects of SCA40, a novel charybdotoxin-sensitive potassium channel opener, in guinea-pig isolated trachealis. Br. J. Pharmacol., 108 (3), 622–6.

2.     Miller C, Moczydlowski E, LaTorre R and Phillips M (1985). Charybdotoxin, a protein inhibitor of single Ca2+-activated K+ channels from mammalian skeletal muscle. Nature (Lond), 313, 316-318.

3.     Book: Guidebook to protein toxins and their use in cell biology, Rappuoli R, Montecucco C, 143-145.

4.     Rauer H, Lanigan MD, Pennington MW, Aiyar J, Ghanshani S, Cahalan MD, Norton RS, Chandy KG (2000). Structure-guided transformation of charybdotoxin yields an analog that selectively targets Ca(2+)-activated over voltage-gated K(+) channels. J Biol Chem, 275(2), 1201-8.

5.     Naranjo D, Miller C (1996). A strongly interacting pair of residues on the contact surface of charybdotoxin and a Shaker K+ channel. Neuron, 16 (1), 123–30.

6.     MacKinnon R, Reinhart PH, White MM (1988). Charybdotoxin block of Shaker K+ channels suggests that different types of K+ channels share common structural features. Neuron, (10): 997–1001.

7.     Garcia ML, Knaus HG, Munujos P, Slaughter RS, Kaczorowski GJ (1995). Charybdotoxin and its effects on potassium channels. Am J Physiol., 269(1.1), C1-10.

8.     Natu VS, Murthy RK, Deodhar KP (2006). Efficacy of species specific anti-scorpion venom serum (AScVS) against severe, serious scorpion stings (Mesobuthus tamulus concanesis Pocock)—an experience from rural hospital in western Maharashtra. J Assoc Physicians India, 54, 283–7.

多肽Pyr-Phe-Thr-Asn-Val-Ser-Cys-Thr-Thr-Ser-Lys-Glu-Cys-Trp-Ser-Val-Cys-Gln-Arg-Leu-His-Asn-Thr-Ser-Arg-Gly-Lys-Cys-Met-Asn-Lys-Glu-Cys-Arg-Cys-Tyr-Ser-COOH的合成步骤：

1、合成CTC树脂：称取0.79g CTC Resin（如初始取代度约为0.98mmol/g）和0.93mmol Fmoc-Ser(tBu)-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2.32mmol 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-Ser(tBu)-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-Ser(tBu)-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2.32mmol Fmoc-Tyr(tBu)-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4.65mmol DIPEA，2.21mmol 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-Tyr(tBu)-Ser(tBu)-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-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Thr(tBu)-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Thr(tBu)-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Cys(Trt)-Thr(tBu)-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Cys(Trt)-Thr(tBu)-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Ser(tBu)-Cys(Trt)-Thr(tBu)-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Ser(tBu)-Cys(Trt)-Thr(tBu)-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Val-Ser(tBu)-Cys(Trt)-Thr(tBu)-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Val-Ser(tBu)-Cys(Trt)-Thr(tBu)-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Asn(Trt)-Val-Ser(tBu)-Cys(Trt)-Thr(tBu)-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Asn(Trt)-Val-Ser(tBu)-Cys(Trt)-Thr(tBu)-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Thr(tBu)-Asn(Trt)-Val-Ser(tBu)-Cys(Trt)-Thr(tBu)-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

H2N-Thr(tBu)-Asn(Trt)-Val-Ser(tBu)-Cys(Trt)-Thr(tBu)-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin

Fmoc-Phe-Thr(tBu)-Asn(Trt)-Val-Ser(tBu)-Cys(Trt)-Thr(tBu)-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-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-Phe-Thr(tBu)-Asn(Trt)-Val-Ser(tBu)-Cys(Trt)-Thr(tBu)-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTC Resin，结构如下：

5、焦谷氨酸反应连接：在上述树脂中，加入适当DMF后，再加入2.32mmol 焦谷氨酸到树脂中，再加入4.65mmol DIPEA、2.21mmol HBTU，鼓氮气反应30分钟。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Pyr-Phe-Thr(tBu)-Asn(Trt)-Val-Ser(tBu)-Cys(Trt)-Thr(tBu)-Thr(tBu)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Trp(Boc)-Ser(tBu)-Val-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Leu-His(Trt)-Asn(Trt)-Thr(tBu)-Ser(tBu)-Arg(Pbf)-Gly-Lys(Boc)-Cys(Trt)-Met-Asn(Trt)-Lys(Boc)-Glu(OtBu)-Cys(Trt)-Arg(Pbf)-Cys(Trt)-Tyr(tBu)-Ser(tBu)-CTCResin。 结构如下：

6、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Pyr-Phe-Thr-Asn-Val-Ser-Cys-Thr-Thr-Ser-Lys-Glu-Cys-Trp-Ser-Val-Cys-Gln-Arg-Leu-His-Asn-Thr-Ser-Arg-Gly-Lys-Cys-Met-Asn-Lys-Glu-Cys-Arg-Cys-Tyr-Ser-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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