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This bombesin receptor antagonist interacts only with the bombesin receptor. Because of this specificity, (D-Phe¹²)-Bombesin may be useful for defining the role of bombesin in various physiological or pathological processes.

Definition

Bombesin is a polypeptide that is found in the brain and gastrointestinal tract. Experimentally it has shown to cause the secretion of various substances (as gastrin and cholecystokinin) and to inhibit intestinal motility.

Discovery

Bombesin was isolated from the skin of the frog Bombina bonmina by Anastasi and team in 19711.

Classification

Bombesin-like peptides are grouped into three families -  1) Bombesin group, viz., bombesin and alytesin, 2) Ranatensin group viz., ranatensins, litorin, and Rohdei litorin, 3) Phyllolitorin group viz., Leu(8)- and Phe(8)-phyllolitorins.

Structural characteristics

Bombesin is a small peptide of 14 amino acids.

Mode of action

The biological activity of bombesin is mediated by binding to specific receptors viz., gastrin releasing peptide receptor (GRPR; called also BB2 receptor), neuromedin B receptor (NMBR; called also BB1 receptor) and bombesin receptor subtype 3 (BRS-3; called also BB3 receptor). Bombesin induce Ca2+ release from intracellular stores2.

Functions

Bombesin and bombesin-like factors show a wide spectrum of biological activities. It regulates the contraction of smooth muscle cells, induction of the secretion of neuropeptides and hormones. It is one of the most powerful substances showing anorexic effects in the hypothalamus. It induces the release of gastrin and cholecystokinin in the intestines and the pancreas. They also posses activities of cytokines.

References

1.     Anastasi A, Erspamer V, Bucci M (1971). Isolation and structure of bombesin and alytesin, 2 analogous active peptides from the skin of the European amphibians Bombina and Alytes. Experientia., 27(2):166-167.

2.     Wang JL, Kalyanaraman S, Vivo MD, Gautam N (1996). Bombesin and thrombin affect discrete pools of intracellular calcium through different G-proteins. Biochem J., 320:87-91.

很多蛋白在细胞中非常容易被降解，或被标记，进而被选择性地破坏。但含有部分D型氨基酸的多肽则显示了很强的抵抗蛋白酶降解能力。

多肽Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-DPhe-Leu-Met-NH2的合成步骤：

1、合成MBHA树脂：取若干克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2.47g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2.22mmol Fmoc-Met-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4.45mmol DIPEA，2.11mmol 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-Met-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-Met-Linker-MBHA Resin

Fmoc-Leu-Met-Linker-MBHA Resin

H2N-Leu-Met-Linker-MBHA Resin

Fmoc-DPhe-Leu-Met-Linker-MBHA Resin

H2N-DPhe-Leu-Met-Linker-MBHA Resin

Fmoc-Gly-DPhe-Leu-Met-Linker-MBHA Resin

H2N-Gly-DPhe-Leu-Met-Linker-MBHA Resin

Fmoc-Val-Gly-DPhe-Leu-Met-Linker-MBHA Resin

H2N-Val-Gly-DPhe-Leu-Met-Linker-MBHA Resin

Fmoc-Ala-Val-Gly-DPhe-Leu-Met-Linker-MBHA Resin

H2N-Ala-Val-Gly-DPhe-Leu-Met-Linker-MBHA Resin

Fmoc-Trp(Boc)-Ala-Val-Gly-DPhe-Leu-Met-Linker-MBHA Resin

H2N-Trp(Boc)-Ala-Val-Gly-DPhe-Leu-Met-Linker-MBHA Resin

Fmoc-Gln(Trt)-Trp(Boc)-Ala-Val-Gly-DPhe-Leu-Met-Linker-MBHA Resin

H2N-Gln(Trt)-Trp(Boc)-Ala-Val-Gly-DPhe-Leu-Met-Linker-MBHA Resin

Fmoc-Asn(Trt)-Gln(Trt)-Trp(Boc)-Ala-Val-Gly-DPhe-Leu-Met-Linker-MBHA Resin

H2N-Asn(Trt)-Gln(Trt)-Trp(Boc)-Ala-Val-Gly-DPhe-Leu-Met-Linker-MBHA Resin

Fmoc-Gly-Asn(Trt)-Gln(Trt)-Trp(Boc)-Ala-Val-Gly-DPhe-Leu-Met-Linker-MBHA Resin

H2N-Gly-Asn(Trt)-Gln(Trt)-Trp(Boc)-Ala-Val-Gly-DPhe-Leu-Met-Linker-MBHA Resin

Fmoc-Leu-Gly-Asn(Trt)-Gln(Trt)-Trp(Boc)-Ala-Val-Gly-DPhe-Leu-Met-Linker-MBHA Resin

H2N-Leu-Gly-Asn(Trt)-Gln(Trt)-Trp(Boc)-Ala-Val-Gly-DPhe-Leu-Met-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Leu-Gly-Asn(Trt)-Gln(Trt)-Trp(Boc)-Ala-Val-Gly-DPhe-Leu-Met-Linker-MBHA Resin

H2N-Arg(Pbf)-Leu-Gly-Asn(Trt)-Gln(Trt)-Trp(Boc)-Ala-Val-Gly-DPhe-Leu-Met-Linker-MBHA Resin

Fmoc-Gln(Trt)-Arg(Pbf)-Leu-Gly-Asn(Trt)-Gln(Trt)-Trp(Boc)-Ala-Val-Gly-DPhe-Leu-Met-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-Gln(Trt)-Arg(Pbf)-Leu-Gly-Asn(Trt)-Gln(Trt)-Trp(Boc)-Ala-Val-Gly-DPhe-Leu-Met-Linker-MBHA 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2.22mmol 焦谷氨酸到树脂中，再加入4.45mmol DIPEA、2.11mmol 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-Gln(Trt)-Arg(Pbf)-Leu-Gly-Asn(Trt)-Gln(Trt)-Trp(Boc)-Ala-Val-Gly-DPhe-Leu-Met-Linker-MBHAResin。 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Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-DPhe-Leu-Met-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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