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This Gastrin I analog is as active as the native sequence, but more stable. The activity loss of Gastrin due to Met sulfoxide formation is avoided.

胃泌素(Gastrin)的定义

胃泌素是胃酸分泌的主要生理调节剂。它对胃粘膜也有重要的营养或生长促进作用。

Gastrin is a major physiological regulator of gastric acid secretion. It also has an important trophic or growth-promoting influence on the gastric mucosa.

胃泌素(Gastrin)的相关肽

前胃泌素在胃窦G细胞中合成并加工成许多生物活性肽。七肽胃泌素-17是主要产物（胃泌素成分3或少量胃泌素），也称为胆囊收缩素B【1】。胃泌素-17酰胺对应于前胃泌素-（55-71）。甘氨酸延伸的非硫酸化胃泌素-17对应于前胃泌素-（55-72）。胃泌素-17的一小部分在G细胞中被切割并作为短COOH末端肽释放，作为胃泌素-7，胃泌素-6和胃泌素-5的混合物【2】。硫酸化胃泌素-6是释放到窦静脉血中的主要形式。胃泌素6比胃泌素17具有更高的效力但功效更低。酪氨酸O-硫酸化增加了胃泌素-6的功效，这也增强了对消除的保护作用。隐球菌素对应于前胃泌素-（1-35）。隐球菌素-（6-35）（前胃泌素-（6-35））是该肽的较短形式【3】。两种最大的α-羧酰胺化胃泌素前体产物是胃泌素71和胃泌素52。胃泌素-52具有生物活性，其功效接近或类似于胃泌素-17【4】。胃泌素成分1是胃泌素的最大激素活性形式，并且已显示对应于胃泌素71。胃泌素-14被称为胃泌素成分4或微胃泌素【5】。五肽胃泌素对应于胃泌素的五个C末端氨基酸，与CCK-5（胆囊收缩素-5）相同，CCK-5主要通过B型胆囊收缩素受体起作用。

Progastrin is synthesized in antral G cells and processed into a number of bioactive peptides. The heptadecapeptide gastrin-17 is the main product (Gastrin component 3 or little Gastrin), called also Cholecystokinin B【1】. Gastrin-17 amide corresponds to Progastrin-(55-71). Glycine-extended nonsulfated gastrin-17 corresponds to Progastrin-(55-72).  A minor fraction of gastrin-17 is cleaved in G cells and released as short COOH-terminal peptides, as a mixture of gastrin-7, gastrin-6, and gastrin-5【2】. The sulfated gastrin-6 is the predominant form released to antral venous blood. Gastrin 6 has a higher potency but a lower efficacy than gastrin-17. The efficacy of gastrin-6 is increased by tyrosine O-sulfation, which also enhances the protection against elimination. Cryptagastrin corresponds to progastrin-(1-35). Cryptagastrin-(6-35) (progastrin-(6-35) is a shorter form of this peptide【3】. The two largest alpha-carboxyamidated progastrin products are gastrin-71 and gastrin-52. Gastrin-52 is bioactive with an efficacy close to or similar to that of gastrin-17【4】. Gastrin component 1 is the largest hormonally active form of gastrin and has been shown to correspond to gastrin-71. Gastrin-14 has been termed gastrin component 4 or minigastrin【5】. Pentagastrin corresponds the five C-terminal amino acids of gastrin and is the same as CCK-5 [cholecystokinin-5], which acts mainly through the type B cholecystokinin receptor.

胃泌素(Gastrin)的发现

胃泌素最初由Edkins和Cantab于1905【6】年鉴定，是胃窦中产生的一种刺激胃酸分泌的因子。它是从猪胃窦粘膜中纯化出来的，并于1964年由Gregory和Tracy测序【7】。

Gastrin was identified originally by Edkins and Cantab in 1905【6】, as a factor produced in the antrum of the stomach that stimulates gastric acid secretion. It was purified from hog antral mucosa and sequenced by Gregory and Tracy in 1964【7】.

 
胃泌素(Gastrin)的结构特征

胃泌素是一种线性肽，合成为前胃泌素，前胃泌素，在胃窦G细胞中加工成许多不同长度的生物活性胃泌素，它们具有相同的含有活性位点的α-酰胺化C末端。胃泌素的活性位点是羧基末端四肽酰胺Trp-Met-Asp-Phe-NH2，所有生物活性片段都具有羧基末端六序列Tyr（SO4）-Gly-Trp-Met-Asp-Phe-NH2。氨基末端延伸的长度控制循环激素形式的代谢和清除。所有生物活性胃泌素肽都是羧酰胺化的，以非硫酸化和硫酸化形式存在【8】。

Gastrin is a linear peptide that is synthesized as a preprohormone, Progastrin, which is processed in antral G cells to a number of bioactive gastrins of different length, which share the same alpha-amidated C-terminus containing the active site. The active site of gastrin is the carboxyterminal tetrapeptide amide Trp-Met-Asp-Phe-NH2 and all bioactive fragments have the carboxyterminal hexasequence Tyr (SO4)-Gly-Trp-Met-Asp-Phe-NH2. The lengths of the aminoterminal extensions govern metabolism and clearance of the circulating hormone forms.  All bioactive gastrin peptides are carboxyamidated and exist in nonsulfated and sulfated forms【8】.

胃泌素(Gastrin)的作用机制

胃泌素受体也是结合胆囊收缩素的受体之一，被称为CCK-B受体。它是G蛋白偶联受体家族的成员。胃泌素的结合刺激细胞内Ca++的增加，蛋白激酶C的激活和肌醇磷酸的产生【9】。

The gastrin receptor is also one of the receptors that bind cholecystokinin, and is known as the CCK-B receptor. It is a member of the G protein-coupled receptor family. Binding of gastrin stimulates an increase in intracellular Ca++, activation of protein kinase C, and production of inositiol phosphate【9】.

胃泌素(Gastrin)的功能

胃泌素似乎对胃肠功能至少有两个主要影响：

Gastrin appears to have at least two major effects on gastrointestinal function:

1、刺激胃酸分泌：胃泌素受体存在于壁细胞上，胃泌素与组胺和乙酰胆碱的结合导致这些细胞完全刺激酸分泌。肠嗜铬样（ECL）细胞也携带胃泌素受体，最近的证据表明，该细胞可能是胃泌素调节酸分泌的最重要靶标。胃泌素刺激ECL细胞导致组胺释放，组胺与壁细胞上的H2受体结合是全面分泌酸所必需的。

1. Stimulation of gastric acid secretion: Gastrin receptors are found on parietal cells, and binding of gastrin, along with histamine and acetylcholine, leads to fully-stimulated acid secretion by those cells. Enterochromaffin-like (ECL) cells also bear gastrin receptors, and recent evidence indicates that this cell may be the most important target of gastrin with regard to regulating acid secretion. Stimulation of ECL cells by gastrin leads to histamine release, and histamine binding to H2 receptors on parietal cells is necessary for full-blown acid secretion.

2、促进胃粘膜生长：胃泌素显然具有刺激胃粘膜发育和生长的许多方面的能力。胃泌素治疗可刺激胃粘膜中的DNA，RNA和蛋白质合成，并增加壁细胞的数量。支持这一功能的另一个观察结果是，患有高胃泌素血症（胃泌素异常高的血液水平）的人始终表现出胃粘膜肥大。

2. Promotion of gastric mucosal growth: Gastrin clearly has the ability to stimulate many aspects of mucosal development and growth in the stomach. Treatment with gastrin stimulates DNA, RNA and protein synthesis in gastric mucosa and increases the number of parietal cells. Another observation supporting this function is that humans with hypergastrinemia (abnormally high blood levels of gastrin) consistently show gastric mucosal hypertrophy.

除了壁细胞和ECL细胞靶标外，胃泌素还通过与胆囊收缩素受体结合来刺激胰腺腺泡细胞，并且已经在某些胃平滑肌细胞群上证实了胃泌素受体，这支持了药理学研究，证明了胃泌素在调节胃动力中的作用。

In addition to parietal and ECL cell targets, gastrin also stimulates pancreatic acinar cells via binding to cholecystokinin receptors, and gastrin receptors have been demonstrated on certain populations of gastric smooth muscle cells, supporting pharmacologic studies that demonstrate a role for gastrin in regulating gastric motility.

胃泌素(Gastrin)的相关文献

1. Morley JS, Tracy HJ, Gregory RA (1965). Structure-function relationships in the active C-terminal tetrapeptide sequence of gastrin. Nature, 207, 1356-1359.
2. Rehfeld JF, Hansen CP, Johnsen AH (1995). Postpoly(Glu) cleavage and degradation modified by O-sulfated tyrosine: a novel posttranslational processing mechanism. EMBO Journal, 14, 389-396.
3. Huebner VD, Jiang RL, Lee TD, Legesse K, Walsh JH, Shively JE, Chew P, Azumi T, Reeve JR Jr (1991). Purification and structural characterization of progastrin-derived peptides from a human gastrinoma. J Biol. Chem., 266(19), 12223-12227.
4. Hansen CP, Stadil F, Rehfeld JF (1995). Metabolism and influence of gastrin-52 on gastric acid secretion in humans. Amer. J of Physiol., 269(4 Pt 1), G600-605.
5. Gregory RA, Tracy HJ, Harris JI, Runswick MJ, Moore S, Kenner GW, Ramage R (1979). Minigastrin: corrected structure and synthesis. Hoppe Seylers Zeitschrift für Physiologische Chemie, 360, 73-80.
6. Edkin JS and Cantab MB (1905). On the chemical mechanism of gastric secretion. Proc. of the Royal Society London, 76, 376.
7. Gregory H, Hardy PM, Jones DS, Kenner GW, Sheppard RC (1964). The antral hormone gastrin. Structure of gastrin. Nature, 204, 931-3.
8. Rehfeld JF and Larsson LI (1979). The predominating molecular form of gastrin and cholecystokinin in the gut is a small peptide corresponding to their COOH-terminal tetrapeptide amide. Acta Physiologica Scandinavia, 115, 117-119.
9. Dockray GJ (1999). Topical review. gastrin and gastric epithelial physiology. J Physiol., 518(2), 315-324.

多肽Pyr-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Leu-Asp-Phe-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1.53g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1.38mmol Fmoc-Phe-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2.75mmol DIPEA，1.31mmol 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-Phe-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-Phe-Linker-MBHA Resin

Fmoc-Asp(OtBu)-Phe-Linker-MBHA Resin

H2N-Asp(OtBu)-Phe-Linker-MBHA Resin

Fmoc-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

H2N-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

Fmoc-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

H2N-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

Fmoc-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

H2N-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

Fmoc-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

H2N-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

Fmoc-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

H2N-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

H2N-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

H2N-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

H2N-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

H2N-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

H2N-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

Fmoc-Leu-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

H2N-Leu-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

Fmoc-Trp(Boc)-Leu-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

H2N-Trp(Boc)-Leu-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

Fmoc-Pro-Trp(Boc)-Leu-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

H2N-Pro-Trp(Boc)-Leu-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-Linker-MBHA Resin

Fmoc-Gly-Pro-Trp(Boc)-Leu-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-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-Gly-Pro-Trp(Boc)-Leu-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-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1.38mmol 焦谷氨酸到树脂中，再加入2.75mmol DIPEA、1.31mmol 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-Gly-Pro-Trp(Boc)-Leu-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-Tyr(tBu)-Gly-Trp(Boc)-Leu-Asp(OtBu)-Phe-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-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Leu-Asp-Phe-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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